Process for producing aglycon by using diglycosidase and flavor-improved food containing the aglycon and converting agent to be used in the process

ABSTRACT

A physiologically active substance of aglycon type, in particular, aglycon isoflavone, can be efficiently produced, without resort to any acid/alkali treatment or fermentation and substantially without changing the physical properties of a material, by treating the material with a sufficient amount of diglycosidase for a sufficient period of time at an appropriate temperature and pH so that a physiologically active substance of glycoside type contained in the material can be converted into the physiologically active substance of aglycon type. Moreover, by using diglycosidase and/or a specific enzyme preparation, the aglycon content in a protein or protein-containing food can be increased and the flavor thereof can be improved.

TECHNICAL FIELD

[0001] The present invention relates to a process for producing anaglycon, a process for producing a protein having an increased aglyconcontent or a food containing said protein, a process for producing aflavor-improved protein or a food containing said protein, and a processfor forming an isoflavone in a living body. The invention may beutilized, for example, for producing processed foods, health foods,dietary supplements, and medicaments.

BACKGROUND ART

[0002] Glycosides are chemical substances wherein a saccharide is bondedto a non-saccharide part called aglycon, and are widely present innature.

[0003] Some glycosides are known to exhibit physiological activitythrough decomposition into aglycons by a glycosidase such as glucosidaseproduced by enteric bacteria though the glycosides themselves do notexhibit the physiological activity because of their poor entericabsorption, as phytochemicals (phytogenic functional ingredients) suchas polyphenols having antioxidation action and phytoestrogens having aweak estrogenic action. Thus, in view of preventing andsymptom-alleviating effects of life-style related diseases such ascancer, arteriosclerosis, osteoporosis, and climacteric disorder, anddiseases owing to aging, attention has been focused on soybean proteinconcentrates, soybean-processed materials, and food containing thesoybean protein concentrates.

[0004] Among the phytochemicals, glucosides that contain isoflavones asaglycons as represented by the following general formula (hereinafter,sometimes referred to as isoflavone glucosides) contain aglycons such asdaidzein, genistein, glycitein, and it is revealed from cellular levelinvestigations and epidemiological surveys that they inhibit growth ofbreast cancer and prostatic cancer cells, alleviate arteriosclerosis andosteoporosis, and also alleviate climacteric disorder owing to thefemale hormone-like estrogenic action.

[0005] (wherein R₁ and R₂ each is independently selected from the groupconsisting of H, OH, and OCH₃, and R₃ is selected from the group of H,COCH₃, COCH₂COOH, and COCH₂CH₂COOH)

[0006] However, there is a possibility that a sufficient amount ofglycosidase cannot be produced by enteric bacteria in elderly persons,sick persons, and antibiotics-administered patients, and also aglycosidase is difficult to decompose glycosides modified with acetyl ormalonyl group and disaccharide or trisaccharide glycosides, so that itcannot be expected to absorb a sufficient amount of phytochemicalscontained in soybean protein concentrates and the like.

[0007] Moreover, it is avoided to take foods containingsoybean-processed materials such as soybean protein concentratesespecially in Western countries owing to the distinctive smell andbitterness, and thus some limitation exists as sources of aglyconisoflavones.

[0008] Accordingly, a process for forming phytochemicals efficiently ina living body, and an improvement of flavor of a soybean-processedmaterial rich in aglycon isoflavones, which are highly efficientlyabsorbed and capable of ingesting a sufficient amount of isoflavones bytaking small amount of them, and having less smell and bitternessdistinctive of soybean or of a food containing the soybean-processedmaterial have been desired.

[0009] On the other hand, the method for converting an isoflavoneglucoside into an aglycon isoflavone is known as described inJP-A-10-117792. In this method, a phytogenic protein extract is treatedwith an alkali to convert a modified glucoside isoflavone into aglucoside isoflavone, which is then subjected to a treatment withglycosidase. Such a treatment is carried out because conventionalglycosidase cannot act directly on the modified glucoside isoflavone.Thus, the method is accompanied by the problems of requirement of twosteps, enhancement of bitterness by the alkali-treatment, change inphysical properties and ingredients, formation of by-products, wasteliquid after the alkali-treatment, and the like. Also, glucosidase whichtakes charge of a main part of the action tends to be influenced by freeglucose, so that the kind and concentration of the material used for theproduction may be limited.

[0010] Moreover, JP-A-8-214787 describes a process for converting aglucoside isoflavone into an aglycon isoflavone by fermentation using amicroorganism. However, there are possibilities of decomposing theresulting aglycon isoflavone by the microorganism and of formingunexpected by-products, and therefore many problems may arise at theactual production.

[0011] Furthermore, a method of hydrolysis with an acid such ashydrochloric acid may be a candidate, but the decomposition of proteins,phospholipids, neutral lipids, and other ingredients may occur alongwith formation of by-products because of the severe conditions.Especially, the formation of chlorinated compounds such as MCP(monochloropropanol) and DCP (dichloropropanol) whose carcinogenicityhas been reported cannot be avoided.

[0012] Therefore, an object of the invention is to provide a process forproducing a phytogenic physiologically active substance of aglycon typeefficiently without resort of any acid/alkali treatment or fermentationand substantially without changing the physical properties of amaterial. Moreover, other objects of the invention are to enhance theaglycon content in a protein or a protein-containing food by usingdiglycosidase and/or a specific enzyme preparation and to improve theflavor. These objects and other objects will be further clarified by thefollowing detailed explanations.

DISCLOSURE OF THE INVENTION

[0013] As a result of extensive studies, we have found thatdiglycosidase discovered from origins of various microorganismsefficiently decompose glycosides which are difficult to decompose byconventional glycosidase and also acts even in a living body, and thusaccomplished the invention. Furthermore, we have found that theinvention can be conducted using any diglycosidase from any source.

[0014] Namely, the invention relates to the following.

[0015] (1) A process for producing an aglycon which comprises forming anaglycon by treating, with diglycosidase, a glycoside containing acompound selected from the group consisting of phytoestrogens,polyphenols, isoflavones, biochanin A, formononetin, cumestrol, andlignans as the aglycon.

[0016] (2) The process for producing an aglycon according to claim 1,wherein the aglycon is an isoflavone.

[0017] (3) The process for producing an aglycon as described above,wherein the glycoside containing an isoflavone as the aglycon is one ormore selected from the group consisting of daidzin, genistin, orglycitin and acetyl derivatives, succinyl derivatives, or malonylderivatives thereof.

[0018] (4) The process for producing an aglycon as described above,wherein the diglycosidase is a glucose-tolerant one.

[0019] (5) The process for producing an aglycon as described above,wherein the diglycosidase is diglycosidase produced by Penicilliummulticolor IAM7153.

[0020] (6) A process for producing a protein having an increased aglyconcontent or a food containing the protein, which comprises a step oftreating a protein or protein-containing food with diglycosidase.

[0021] (7) The process for producing a protein having an increasedaglycon content or a food containing the protein as described above,wherein the protein or protein-containing food contains a glycosidecontaining an isoflavone as the aglycon.

[0022] (8) The process for producing a protein having an increasedaglycon content or a food containing the protein as described above,wherein the protein or protein-containing food to be produced is afurther flavor-improved one.

[0023] (9) The process for producing a protein having an increasedaglycon content or a food containing the protein as described above,wherein the glycoside containing an isoflavone as the aglycon is one ormore selected from the group consisting of daidzin, genistin, orglycitin and acetyl derivatives, succinyl derivatives, or malonylderivatives thereof.

[0024] (10) The process for producing a protein having an increasedaglycon content or a food containing the protein as described above,which further comprises a step of treating with an enzyme preparationcontaining mainly at least one enzyme selected from the group consistingof amylases, proteases, lipases, α-glucosidases, and yeast-dissolvingenzymes.

[0025] (11) The process for producing a protein having an increasedaglycon content or a food containing the protein as described above,wherein the improvement of flavor is reduction of bitterness and/orastringency.

[0026] (12) A process for producing a flavor-improved protein or a foodcontaining the protein, which comprises a step of treating with anenzyme preparation containing mainly at least one enzyme selected fromthe group consisting of amylases, cellulases, pectinases, proteases,lipases, a-glucosidases, a-galactosidase, and yeast-dissolving enzymes.

[0027] (13) The process for producing a flavor-improved protein or afood containing the protein as described above, wherein the protein orprotein-containing food contains a glycoside containing a flavonoid asthe aglycon.

[0028] (14) The process for producing a flavor-improved protein or afood containing the protein as described above, wherein the protein orprotein-containing food contains a glycoside containing an isoflavone asthe aglycon.

[0029] (15) A method of administering diglycosidase orally to form anaglycon from a glycoside in a living body.

[0030] (16) The method as described above, wherein diglycosidase isorally administered to form an aglycon in a living body from a glycosidecontaining an isoflavone as the aglycon.

[0031] (17) A method of converting a physiologically active substance ofglycoside type into a physiologically active substance of aglycon type,which comprises treating the physiologically active substance ofglycoside type with diglycosidase.

[0032] (18) A process for producing a composition rich in a phytogenicphysiologically active substance of aglycon type, which comprisestreating a phytogenic material containing a phytogenic physiologicallyactive substance of glycoside type with diglycosidase.

[0033] (19) A method of accelerating a bioabsorption of aphysiologically active substance, which comprises administeringdiglycosidase orally before, during, or after the ingestion of a foodcontaining a physiologically active substance of glycoside type.

[0034] (20) An agent converting a physiologically active substance ofglycoside type into the physiologically active substance of aglycontype, which contains at least diglycosidase.

[0035] These embodiments and other embodiments of the invention will befurther clarified by the following detailed explanations.

[0036] Diglycosidase described in the invention efficiently acts on theglycosides that contain a compound selected from the group consisting ofphytoestrogens, polyphenols, isoflavones, biochanin A, formononetin,cumestrol, and lignans as the aglycon (hereinafter, also referred to asaglycon glycoside), can very efficiently act especially on theglycosides containing an isoflavone as the aglycon (hereinafter, alsoreferred to as isoflavone glycosides), and is hardly influenced by freeglucose. Therefore, the process can be advantageously carried out in thecase that the isoflavone glycoside is daidzin, genistin, or glycitin, oran acetyl derivative, succinyl derivative, or malonyl derivativethereof. By the way, the isoflavone formed from the isoflavone glycosideis also referred to as aglycon isoflavone.

[0037] Moreover, by administering the diglycosidase, the preventiveeffect on various diseases can be enhanced through the formation of anisoflavone from an aglycon glycoside or a food containing the same whichis orally ingested. Diglycosidase produced by Penicillium multicolorIAM7153 is preferably used as diglycosidase. β-Galactosidase derivedfrom Penicillium multicolor is an enzyme which is described in FoodAdditives List and whose safety is recognized, and thus diglycosidaseproduced by such highly safety bacterium is estimated to be highly safe.

[0038] Furthermore, in the case of using soybean as a starting material,although the benefit of soybean protein concentrates andsoybean-processed materials to health is reported, ingestion of them areavoided especially in Western countries owing to the distinctive smelland bitterness. The above-mentioned acetylglycoside isoflavones andmalonylglycoside isoflavones have a strong bitterness but the aglyconisoflavones have less bitterness. Therefore, a food wherein bitternessof soybean protein concentrate or soybean-processed material isefficiently reduced can be provided by the conversion into aglyconisoflavones by diglycosidase.

[0039] When such soybean protein concentrate or soybean-processedmaterial wherein isoflavones are concentrated as aglycons having higherabsorption efficiency is used, a sufficient amount of isoflavones can beingested through a little intake, and also the form can be changed to aform easily ingested by people who avoid the smell and strange tastedistinctive of soybean.

[0040] Moreover, owing to the smell and strange taste of soybean proteinconcentrates or soybean-processed materials, the amount used is limitedin the case of using them as food materials, and therefore soybeanprotein concentrates or soybean-processed materials have a limitation asisoflavone sources for foods. According to the invention, use of thesoybean protein concentrates or soybean-processed materials, whereinisoflavone glycosides are digested with diglycosidase and concentratedas isoflavone aglycons which have higher absorption efficiency and lessbitterness, enables supply of isoflavones to foods through a little useof the concentrates or materials, so that they can be utilized for manykinds of foods as isoflavone sources.

[0041] Diglycosidase for use in the invention is characterized in thatit has an activity of acting on a disaccharide glycoside, which isdifficult to utilize as a substrate by conventional glucosidase, toisolate a saccharide as a two-saccharide unit from the disaccharideglycoside and also to form an aglycon. Herein, an enzyme having theabove activity is referred to as “diglycosidase”.

[0042] The diglycosidase in the invention is an enzyme which isclassified into a saccharide-chain hydrolase but has a propertydifferent from the properties of conventional α- and β-glycosidases.Diglycosidase can utilize, as a substrate, so-called a glycoside whereina linear or branched saccharide chain composed of single or two or morekinds of saccharides is bonded to a compound other than a saccharidethrough hydroxyl group in the saccharide chain, and recognizes thesubstrate at the two-saccharide unit to cleavage it, wherebycorresponding disaccharide and an aglycon having a saccharide chain withtwo saccharide-smaller chain length are formed successively and finally,an aglycon is formed. Additionally, it also decomposes modifiedglucosides such as acetyl derivatives, succinyl derivatives, and malonylderivatives, which are difficult to decompose by conventionalglucosidase, into saccharides and aglycons. As representative examplesof saccharides present in nature, starch, cellulose, polysaccharidesconstituting cell walls, and the like may be mentioned. Many kinds ofsaccharide chains may be suitable for the saccharide chains ofglycosides, and examples thereof include6-O-β-D-xylopyranosyl-β-D-glucopyranoside (β-primeveroside),6-O-α-L-arabinopyranosyl-β-D-glucopyranoside (vicianoside),6-O-α-L-arabinofuranosyl-β-D-glucopyranoside,6-O-α-L-rhamnopyranosyl-β-D-glucopyranoside (rutinoside),6-O-β-D-apiofuranosyl-β-D-glucopyranoside,6-O-β-D-glucopyranosyl-β-D-glucopyranoside (gentiobioside),4-O-α-glucopyranosyl-β-D-glucopyranoside (maltose),2-O-α-L-rhamnopyranosyl-β-D-galactopyranoside (rhaminose),6-O-α-L-rhamnopyranosyl-β-D-galactopyranoside (robinobioside),2-O-β-D-xylopyranosyl-β-D-glucopyranoside (xylosylglucose),4-O-β-D-glucopyranosyl-β-D-glucopyranoside (cellobioside), xylobioside,and the like. Other than the above-mentioned compounds, any combinationof saccharides can be recognized as a substrate for the reaction as faras the combination has a disaccharide structure. Aglycon means acompound to be obtained from a glycoside by eliminating a saccharide ofthe glycoside. Aglycons of glycosides are widely present in nature, andexamples thereof include volatile compounds in plants such as linalool,geraniol, citronellal, phenethyl alcohol, citronellol, jasmones,limonene, terpinene, citral, nerol, pinene, borneol, terpineol, methyljasmonate, hexanol, hexenol, hexanal, hexenal, vanillin, benzaldehyde,eugenol, methyl salicylate, linalool oxide, benzyl alcohol, andvomifomitol; pigments in plants such as alizarin, purpurin,anthocyanidin including pellagonidin, cyanidin, delphinidin, peonidin,petunidin, and malvidin; and flavonoids such as nariltin, naringenin,hesperetin, neohesperetin, diosmetin, quercetin, campherol, myricetin,isorhamnetin, and syringenin; and the like. Other than the compoundsmentioned herein, various compounds may be present as aglycons ofglycosides or may become aglycons of glycosides.

[0043] Furthermore, diglycosidase can utilize so-called monosaccharideglycosides, wherein one molecule of saccharide is bonded to an aglycon,as substrates to form corresponding monosaccharides and aglycons, otherthan above-mentioned disaccharide-isolating activity. In particular, itis a characteristic that diglycosidase can act on monosaccharideglycosides which is resistant to hydrolysis by conventionalβ-glucosidase.

[0044] Diglycosidase for use in the invention can be obtained frommicroorganisms having ability of producing diglycosidase withoutrequiring undue experimental burden from those skilled in the art (Forexample, cf. WO00/18931).

[0045] The microorganisms producing diglycosidase of the invention canbe obtained by the following screening, for example. That is, anenrichment culture is carried out by inoculating a soil suspension to aliquid medium for separation containing eugenyl primeveroside or thelike as sole carbon source, applying the culture liquid onto a similarplating agar medium for separation and selecting colonies grown. Thesestrains are cultured in a suitable liquid medium and strains havingpNP-isolating activity can be selected through cleavage of disaccharidefrom pNP-primeveroside or the like.

[0046] On these strains thus selected, microorganisms producingdiglycosidase can be screened using pNP-primeveroside or the like as asubstrate and disaccharide isolation as a measure.

[0047] The producing ability has been already confirmed on Aspergillusniger IFO4407 (available from Institute of Fermentation, 2-17-85,Juso-honmachi, Yodogawa-ku, Osaka), Aspergillus niger IAM 2020,Aspergillus fumigatus IAM2046, Penicillium multicolor IAM7153 (availablefrom Institute of Molecular Cell Biology, the University of Tokyo,1-1-1, Yayoi, Bunkyo-ku, Tokyo), and the like.

[0048] Additionally, in other various microorganisms, the diglycosidaseactivity has been confirmed on various microorganisms such as the genusAspergillus, the genus Penicillium, the genus Rhizopus, the genusRhizomucor, the genus Talaromyces, the genus Mortierella, the genusCryptococcus, the genus Microbacterium, the genus Corynebacterium, thegenus Actinoplanes, and the like.

[0049] Any strain can be used in the invention as far as it has anability of producing diglycosidase, and the strain is not limited to theabove-mentioned strains. Furthermore, the process for producingdiglycosidase usable in the invention includes mutant strains of thestrains having a diglycosidase-producing ability, or variousmicroorganisms or various cells (e.g., yeast cells, bacterial cells,higher plant cells, and animal cells) modified so as to be capable ofproducing diglycosidase by recombinant DNA method, and particularlypreferred are those modified so as to be capable of producingdiglycosidase with high productivity. In the case that adiglycosidase-producing ability is imparted by introducing adiglycosidase gene, the microorganism used as a host may not have adiglycosidase-producing ability.

[0050] For producing diglycosidase using the above variousmicroorganisms, a method and conditions suitable for the culture of themicroorganism can be set, and the method and conditions are notparticularly limited. For example, any of liquid culture and solidculture may be used for culturing the above various strains, but liquidculture is preferably used. The liquid culture may be carried out asfollows, for example.

[0051] The medium to be employed may be any medium as far as themicroorganism producing diglycosidase is capable of growing in themedium. For example, there may be used media to which carbon sourcessuch as glucose, sucrose, gentiobiose, soluble starch, glycerol,dextrin, molasses, and organic acids; further nitrogen sources such asammonium sulfate, ammonium carbonate, ammonium phosphonate, ammoniumacetate, or peptone, yeast extract, corn steep liquor, caseinhydrolysate, bran, and meat extract; and further inorganic salts such aspotassium salts, magnesium salts, sodium salts, phosphonates, manganesesalts, iron salts, and zinc salts are added. Furthermore, foraccumulating diglycosidase, various inducing substances may be added tothe medium. As the inducing substances, saccharides may be used, forexample, and there may be preferably used gentose (e.g., gentose #80,Nihon Shokuhin Kako Co., Ltd.), gentiobiose, genti-oligosaccharide(e.g., gentiologo etc., Wako Pure Chemical Industries, Ltd.),galactomannan, and the like. The adding amount of these inducingsubstances is not particularly limited as far as the productivity ofaimed diglycosidase is enhanced, but the substance is preferably addedin an amount of 0.01 to 10%.

[0052] The pH of the medium is adjusted to from about 3 to 8, preferablyfrom about 5 to 6, and culture is carried out at a temperature of about10 to 50° C., preferably about 25 to 30° C. for 1 to 15 days, preferably4 to 7 days under aerobic conditions. As the culturing method, a shakingculture or an aerobic submerged culture by means of a jar fermenter maybe utilized. However, the above various culturing conditions may beoptionally changed, of course, depending on the microorganism or cell tobe cultured, and the conditions are not particularly limited as far asdiglycosidase of the invention is produced.

[0053] For isolation and purification of diglycosidase from the cultureliquid obtained, using a diglycosidase activity as a measure, purifieddiglycosidase can be obtained by combining centrifugal separation, UFconcentration, salting out, and various chromatography such as ionexchange resins, and treating in a usual manner (Referential document:Tanpakusitsu·Kouso no Kisojikkenhou (Basic experimental methods forproteins and enzymes), written by Takekazu Horio, Nankodo).

[0054] A culture liquid obtained by culturing the above microorganismmay be utilized as such as the enzyme composition of the invention. Ofcourse, the culture liquid may be optionally changed in the degree ofpurification according to the purpose used in the invention.

[0055] The invention provides a process for producing an aglycon whichcomprises forming an aglycon by treating, with diglycosidase, aglycoside containing a compound selected from the group consisting ofphytoestrogens, polyphenols, isoflavones, biochanin A, formononetin,cumestrol, and lignans as the aglycon. The producing process includesthe reaction of a phytogenic material containing the above compound asthe aglycon with a sufficient amount of diglycosidase under weaklyacidic conditions at an appropriate temperature and pH for a sufficientperiod of time so as to convert at least most of the glycoside in thestarting material into an aglycon, whereby an aglycon is produced. Theinvention provides a producing process wherein diglycosidase is added toa plant extract in order to produce a plant extract rich in an aglycon.

[0056] The novel process is a one-step process of converting most of anaglycon glycoside into free aglycon by an enzyme preparation containinga hydrolase of disaccharide glycosides, i.e., diglycosidase. The processis effective for the aglycon glycosides present in phytogenic materials,preferably proteins or protein foods. Since the process is found to becapable of substantially complete conversion of modified glucosideisoflavones and glucoside isoflavones into aglycon isoflavones, itincludes the conversion of modified glucoside isoflavones and glucosideisoflavones into aglycon isoflavones. In some phytogenic proteinmaterials, particularly soybean protein materials, substantial part oftotal isoflavone contents in the phytogenic protein materials is presentin the form of isoflavone glycosides. Therefore, not only the conversionof glycoside isoflavones into aglycon isoflavones but also theconversion of modified glycoside isoflavones into aglycon isoflavonesare necessary for maximum increase of the amount of aglycon isoflavonesobtainable from the phytogenic protein materials.

[0057] The starting material in a preferred embodiment is any protein orprotein-containing food (more preferably a phytogenic material,phytogenic protein, or phytogenic protein-containing food) containing aphysiologically active substance of glycoside type. Some processes inthe following explanations are described using soybean products asexamples, but the process of the invention can be generally applied to awide range of proteins or protein-containing foods other than soybeanand soybean products.

[0058] In the invention, the “protein or protein-containing food”preferably contains a physiologically active substance of glycoside typebut is not particularly limited.

[0059] The “phytogenic material” in the invention means a whole plantbody which is edible or used as a medicine, or a part thereof such asleaf, flower, fruit, stem, or root, or a processed product thereof.Examples thereof include whole plant bodies harvested, or parts thereofsuch as leaf, flower, fruit, stem, and root, and plant extracts andprocessed products thereof. Specific examples of the phytogenic materialinclude the following materials: phytogenic proteins such as soybeanprotein, soymilk, juices (orange juice, grape juice, apple juice,pomegranate juice), herb tea, plant extracts such as herb extract, andprocessed products of the above materials such as juice drinks, wine,tea, black tea, and cocoa.

[0060] The “phytogenic protein” means a protein obtainable from theabove “phytogenic material”, and may be a mixture with other ingredientsderived from the phytogenic material.

[0061] In the invention, the “compound selected from the groupconsisting of phytochemicals, phytoestrogens, polyphenols, isoflavone,biochanin A, formononetin, cumestrol, and lignans” is not particularlylimited as far as the compound falls within these conceptual range, butit is preferably a compound which exhibits a physiological activity orenhances a physiological activity in a living body (preferably awarm-blooded animal, more preferably human). The compound is preferablya flavonoid, more preferably an isoflavone, most preferably anisoflavone represented by the above structural formula.

[0062] The “physiologically active substance” in the invention means asubstance, most of which is preferably present as a glycoside in a plantbody and which exhibits a physiological activity or enhances aphysiological activity in a living body upon the conversion into theaglycon type. Specifically, the physiologically active substanceincludes phytochemicals, phytoestrogens, polyphenols, isoflavone,biochanin A, formononetin, cumestrol, and lignans as mentioned above,and preferred are isoflavones.

[0063] The “physiologically active substance of glycoside type” meansthat the aglycon of the above glycoside is a physiologically activesubstance, and the saccharide chain is composed of one or moresaccharide, preferably two or more saccharides. The two-saccharidechains include those mentioned above and the like.

[0064] The “enzyme preparation containing mainly at least one enzymeselected from the group consisting of amylases, proteases, lipases,α-glucosidases, and yeast-dissolving enzymes” is not particularlylimited as far as it mainly contains these enzymes, and commerciallyavailable enzymes may be employed. The following will illustrates thosemanufactured by Amano Enzyme Inc. Examples of amylase include Amylase AD“Amano” 1 (optimum pH: 6.0, optimum temperature: 70° C.), Gluczyme NL4.2 (optimum pH: 4.5, optimum temperature: 65° C.), Transglucosidase L“Amano” (optimum pH: 5.0, optimum temperature: 60° C.), and the like.Examples of cellulase include Cellulase A “Amano” 3 (optimum pH: 4.5,optimum temperature: 55° C.), Cellulase T “Amano” 4, Hemicellulase“Amano” 90G (optimum pH: 4.5, optimum temperature: 50° C.),Hemicellulase GM “Amano”, and the like. Examples of pectinase includePectinase PL “Amano” (optimum pH: 4.55-0, optimum temperature: 60-55°C.) and the like. Examples of protease include Umamizyme, Newlase F3G,Papain W-40, Pancreatin F, Protease B, Protease A “Amano” G. and thelike, and examples of lipase include Lipase A “Amano” 6 (optimum pH:6.5, optimum temperature: 45° C.), and the like. A yeast-dissolvingenzyme preparation YL-15 (optimum pH: 7.0, optimum temperature: 50-55°C.) is mentioned as a yeast-dissolving enzyme, and ADG-S-DS (optimum pH:4.5-5, optimum temperature: 50-60° C.) and the like are mentioned as anα-galactosidase.

[0065] The above enzymes and enzyme preparations can be produced byknown methods. For example, an enzyme can be obtained by screening amicroorganism producing a specific enzyme mentioned above in a similarmanner to the production of diglycosidase and culturing the resultingenzyme-producing strain in a suitable medium. Examples of the aboveenzyme-producing strain include Bacillus subtillis, Aspergillus niger,Aspergillus oxyzae, Trichoderma viride, Rhizopus nivenus, Pseudomonassp., and the like.

[0066] The following will explain the invention in further detail withregard to the process for producing a protein having an increasedaglycon content or a food containing the protein, which comprises a stepof treating a protein or protein-containing food with diglycosidase, byway of illustration of a phytogenic physiologically active ingredient(especially an isoflavone glycoside) derived from a phytogenic material,but the invention can be conducted using any above compound other thanthe phytogenic physiologically active ingredient derived from aphytogenic material. By the way, the term of soybean material usedherein means any type of soybean or variants of soybeans.

[0067] Some different embodiments are possible as specific processes forcarrying out the invention.

[0068] In the first embodiment, a phytogenic physiologically activesubstance of glycoside type is converted into a phytogenicphysiologically active substance of aglycon type while the phytogenicphysiologically active substance is left in the phytogenic material.Therefore, the formed phytogenic physiologically active substance ofaglycon type may be left in the phytogenic material or may be suitablyremoved. The aglycon form of the phytogenic physiologically activesubstance may be generally removed by a solvent, hydrophobic effluenceor extraction. The solvent suitable for the operation includes acetone,ethanol, and other similar organic solvents, but is not limited thereto.

[0069] In the second embodiment, a phytogenic physiologically activesubstance of glycoside type (e.g., isoflavone modified glycoside orisoflavone glucoside) in a phytogenic material is removed from thephytogenic material by aqueous effluence or extraction. The aqueouseffluence is carried out through the effluence of relatively solublephytogenic physiologically active substance of glycoside type byimmersing the phytogenic material or by exposing the phytogenic materialto or dipping it in water or a mixture of hydrophilic solvents such asethanol or other alcohols. The pH of the resulting aqueous solution isfrom about pH 2 to about pH 5, preferably about pH 4. After removal, thephytogenic physiologically active substance of glycoside type isconverted into the phytogenic physiologically active substance ofaglycon type.

[0070] In the third embodiment, prior to all the operations forconversion, a phytogenic physiologically active substance of glycosidetype is removed from a phytogenic material.

[0071] Depending on the type of phytogenic material containing aphytogenic physiologically active substance of glycoside type, in somecases, the phytogenic material is preferably processed to a finelycrushed form. This operation is desirable for bringing a phytogenicphysiologically active substance in the phytogenic material into contactwith a reagent (diglycosidase) employed in the step which will bedescribed in detail in the following. The material may be subjected togrinding, crushing, or other processing. When the phytogenic material isin condition that isoflavone compounds in the phytogenic material easilycome into contact with an external reagent or reactant, e.g., a smallleaf part in a plant, it is not necessary to subject the phytogenicmaterial to the above processing.

[0072] The conversion of a phytogenic physiologically active substanceof glycoside type into the phytogenic physiologically active substanceof aglycon type is sometimes partially carried out by enzymes present inthe mixture depending on the phytogenic material used. These enzymes maybe present naturally in phytogenic protein materials or may be derivedfrom microorganisms grown in the materials. Such enzymes are called asresidual enzymes. However, there is a possibility that the conversion ofthe phytogenic physiologically active substance of glycoside type intothe phytogenic physiologically active substance of aglycon type cannotbe carried out sufficiently depending on the nature and concentration ofthe residual enzyme in the phytogenic protein materials. By adding anenzyme preparation containing an external enzyme, i.e., diglycosidase,maximum converting efficiency of the phytogenic physiologically activesubstance of aglycon type can be achieved.

[0073] In the invention, the amount of the enzyme to be added depends onvarious factors including the type of enzyme present, the distributionof enzyme concentration, the pH of reaction system, the activity ofenzyme present, and temperature. In the case of adding an enzyme,typically preferred enzyme amount is preferably from 28 to 2800 AU,usually from 10 to 10000 AU relative to 100 g of a phytogenic materialas total concentration of enzyme present based on dry weight thereof.When a sufficient concentration of enzymes including a residual enzyme,an additional enzyme or both enzymes are present in the system, aphytogenic physiologically active substance of glycoside type is broughtinto contact with the enzymes at an appropriate temperature and pH for asufficient period of time so as to convert substantially all thephytogenic physiologically active substance of glycoside type in themixture into the phytogenic physiologically active substance of aglycontype.

[0074] The conversion-production step is preferably carried out at a pHof about 2 to about 6. More preferred pH range for theconversion-production step is from about 3 to about 5. Depending on thephytogenic material used, the pH may be adjusted with an acidic reagentsuch as hydrochloric acid, phosphoric acid, acetic acid, or sulfuricacid, or an alkaline reagent such as sodium hydroxide. In many cases, itis assumed to use an acidic or alkaline reagent of food grade. Thetemperature to be used in the conversion-production step is preferablyfrom about 25° C. to about 65° C. More preferred temperature is fromabout 30° C. to about 55° C. Throughout the reaction, the temperature isusually constant, but the temperature may be elevated or loweredaccording to the successive step and final intended use. Namely, it maybe relatively freely changed according to the various circumstances ofthe situation.

[0075] The period of time necessary for the conversion and productionmay be determined depending on complicated relationship between variousfactors of the kind, concentration, and physical properties of thematerial to be reacted, the concentration of the enzyme added, andfurther the temperature and pH of the reaction system. In most cases,the conversion-production can be substantially completely achievedwithin 6 to 12 hours. The period of time for the conversion-productioncan be shortened depending on the concentration of the diglycosidasepreparation added. At the conversion-production step, most of theisoflavone glycoside in the mixture can be converted into the aglyconisoflavone. The efficiency of the conversion is usually at least about50% or more, preferably about 70% or more. By adopting the abovepreferable reaction conditions, nearly complete conversion can beachieved.

[0076] By adopting conditions similar to the above, it is possible tocarry out a process of the invention for producing an aglycon whichcomprises forming an aglycon by treating, with diglycosidase, aglycoside containing a compound selected from the group consisting ofphytoestrogens, polyphenols, isoflavones, biochanin A, formononetin,cumestrol, and lignans as the aglycon.

[0077] In addition to the above step, in the invention, the process mayfurther comprise a step of treating with an enzyme preparationcontaining mainly at least one enzyme selected from the group consistingof amylases, proteases, lipases, α-glucosidase, and yeast-dissolvingenzymes. This step may be conducted before or after the step of treatingwith diglycosidase, or the treatment with diglycosidase and the enzymepreparation may be carried out at the same time. In this case, thetreatment with diglycosidase and the enzyme preparation at the same timeis carried out under the conditions similar to those in the case ofusing diglycosidase solely. Moreover, when the treatment with an enzymepreparation is carried out before or after the treatment withdiglycosidase, the pH, temperature, period of time, and the like may beselected in consideration of optimum pH and optimum temperature of theabove each enzyme preparation. This process is also accompanied by theeffects of increasing aglycon content in a protein or protein-containingfood and of improving flavor through the reduction of bitterness and/orastringency.

[0078] Additionally, in the invention, during the process of finding theabove effects of the combined use with diglycosidase, it was found thatflavor is improved by treating a protein or protein-containing food witha specific enzyme preparation alone, i.e., an enzyme preparationcontaining mainly at least one enzyme selected from the group consistingof amylases, cellulases, pectinases, proteases, lipases, a-glucosidase,α-galactosidases, and yeast-dissolving enzymes. With regard to thetreating conditions in this case, the pH is preferably from 3 to 8, morepreferably from 5 to 7.5, and the treating temperature and treating timeare similar to the case of the combined use with diglycosidase. By theway, the treated product may be optionally adjusted to a desired pH.

[0079] By treating a protein or protein-containing food as mentionedabove, the flavor of the protein or protein-containing food can beimproved and particularly, bitterness and/or astringency can be reduced.

[0080] In addition, by administering the phytogenic physiologicallyactive substance of aglycon type produced as above or a composition richin the phytogenic physiologically active substance of aglycon type assuch or as a mixture with a food or drink, the effect derived from thephytogenic physiologically active substance can be attained. The effectsof the phytogenic physiologically active substance include effects ofpreventing various diseases (cancer, life-style related diseases,osteoporosis, a burning sensation in climacteric disorder, and thelike), and of regulation of intestinal function, immunostimulation, andbiophylactic action. Moreover, other than the administration of thephytogenic physiologically active substance which is converted intoaglycon type beforehand, by administering orally a phytogenicphysiologically active substance of glycoside type and/or a phytogenicmaterial containing a phytogenic physiologically active substance ofglycoside type together with diglycosidase, the phytogenicphysiologically active substance of glycoside type is converted into thephytogenic physiologically active substance of aglycon type in a livingbody, for example in stomach or intestines and the absorption of thephytogenic physiologically active substance of aglycon type and themigration into blood are accelerated, whereby preventive effect of thephytogenic physiologically active substance to various diseases can beenhanced.

[0081] The method of accelerating a bioabsorption of a physiologicallyactive substance according to the invention, which comprisesadministering diglycosidase orally before, during, and/or after theingestion of a food containing physiologically active substances ofglycoside type, (preferably, a method of forming an isoflavone from aglycoside containing an isoflavone as the aglycon in a living body), isconducted as follows.

[0082] The target is a warm-blooded animal, preferably human orlivestock.

[0083] As far as the phytogenic physiologically active substance ofglycoside type comes into contact with diglycosidase in stomach andintestines, diglycosidase may be administered at any time before,during, or after the ingestion of a food containing physiologicallyactive substances of glycoside type. Preferred is between just after ameal and one hour after the meal.

[0084] The dose of diglycosidase is not particularly limited as far asthe conversion of the phytogenic physiologically active substance ofglycoside type into the phytogenic physiologically active substance ofaglycon type occurs in a living body, but diglycosidase is orallyadministered in an amount of usually from 10 mg/day to 500 mg/day,preferably from 30 mg/day to 300 mg/day, more preferably 100 mg/day to200 mg/day. The number of dose is not particularly limited but ispreferably from once per several days to several times per day,particularly preferably three times per day (i.e., after every meal).

[0085] Moreover, the ingesting amount of the physiologically activesubstance is not particularly limited as far as its effect is attained,but the substance is ingested in an amount of preferably 10 mg/day ormore, more preferably 50 mg/day or more, further preferably 50 mg/day to100 mg/day.

[0086] Furthermore, diglycosidase may be administered solely, as anenzyme preparation, and/or as a mixture with conventional glycosidase(e.g., glucosidase, galactosidase, etc.).

[0087] Diglycosidase may be used as an enzyme preparation. In this case,the enzyme preparation contains diglycosidase as the essentialingredient, and may further contain various enzymes, stabilizers, andthe like.

[0088] Additionally, in the case of the administration as an enzymepreparation mixed with conventional glycosidase, examples of theconventional glycosidase include glucosidase, galactosidase, xylosidase,and rhamnosidase, and the dose of diglycosidase is preferably from 10mg/day to 50 mg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

[0089]FIG. 1 is a graph showing the results of Example 7.

[0090]FIG. 2 is a graph showing the results of Example 8.

[0091]FIG. 3 is a graph showing the results of Example 14.

[0092]FIG. 4 is a graph showing the results of Example 15.

BEST MODE FOR CARRYING OUT THE INVENTION

[0093] The invention will be explained in detail with illustratingExamples using soybean materials as phytogenic materials. Examples areillustrated for the purpose of explanation only and they by no meansrestrict the scope of the invention.

[0094] The phytogenic materials, for example defatted soybean, soymilk,concentrated soybean protein, and various soybean products contain 12kinds of isoflavone compounds. Specifically, they contain aglycons ofglycitein, daidzein, and genistein; glucoside glycosides of glycitin,daidzin, and genistin; acetylglycitin, acetyldaidzin, and acetylgenistinhaving O-acetyl group at 6-position of the glucose residue; andmalonylglycitin, malonyldaidzin, and malonylgenistin having O-malonylgroup at 6-position of the glucose residue. The existing ratio of thesecompounds is characteristic to each of the difference of varieties ofsoybean and the difference of treatment in the production steps.

[0095] Unless otherwise stated, ratio, part(s), percent, and the likeare herein based on weight.

[0096] By the way, the measured activities of various enzymes herein areshown as values obtainable by the method described below unlessotherwise stated.

[0097] Diglycosidase Activity

[0098] The activity was measured on an automatic chemical analyzingapparatus (TBA-30R manufactured by Toshiba Corporation). Thirty μL of anenzyme sample was mixed with 200 μL of 2 mM solution of p-nitrophenyl(pNP) primeveroside used as a substrate of disaccharide glycoside, whichis obtained by dissolving the compound in an acetate buffer (pH 5.5),followed by reaction at 40° C. for 9.75 minutes at the cycle time of22.5 seconds. Then, 250 μL of sodium carbonate was added thereto andthen absorbance at 412 nm was measured. A blank derived from the samplewas measured similarly using 20 mM acetate buffer (pH 5.5) instead ofthe substrate solution.

[0099] The enzyme amount increasing the absorbance by 1 under theconditions is defined as 1 AU.

[0100] The pNP-primeveroside used herein can be synthesized, forexample, by reacting pNP-glucoside (manufactured by Merck) withxylo-origosaccharide (manufactured by Wako Pure Chemical Industries,Ltd.) using an enzyme, xylosidase (manufactured by Sigma) to bond xyloseto pNP-glucoside in β-1,6-manner via one residue transfer.

EXAMPLE 1 Production of Diglycosidase by Penicillium multicolor IAM7153

[0101] Culture of Diglycosidase

[0102] A medium for growth (pH 5.6) containing 2.0% of defatted soybean,3.0% of glucose, 0.5% of potassium dihydrogen phosphate, 0.4% ofammonium sulfate, 0.3% of dry yeast was sterilized at 121° C. for 20minutes. To 100 mL of the sterilized medium was inoculated 1 oese ofPenicillium multicolor IAM7153, followed by pre-culture at 27° C. at theshaking rate of 140 min⁻¹. After 5 days, 20 L of a main medium of pH 4.9containing 1.0% of Sunfiber R, 2.0% of potassium dihydrogen phosphate,1.0% of ammonium sulfate, and 3.13% of meast P1G was sterilized in a 30L jar fermenter at 121° C. for 20 minutes while stirring at 150 min⁻¹.The pre-medium was inoculated at a rate of 1.5% and the whole wascultured at a stirring number of 250 min⁻¹, an aeration of 0.75 vvm (15L/min), an inner pressure of 0.5 kg/cm² (48 kPa), and a temperature of27±1° C. for 8 days.

[0103] Purification of Diglycosidase

[0104] To the culture broth were added 2% by weight each, based on totalliquid amount, of Zemlite Super 56M and Fineflow A as filtration aidsand filtration through diatomaceous earth was carried out. The filtratewas concentrated by a factor of 20 using an ultrafiltration membrane UFAIP-2020 (MW 6,000) and also the substitution by 20 mM acetate buffer ofpH 4.7 was conducted. Ammonium sulfate was added to the aboveultrafiltration concentrate to conduct 50% ammonium sulfate-salting out.The resulting precipitate was removed and ammonium sulfate was furtheradded to the supernatant to conduct 80% ammonium sulfate-salting out.The precipitate was recovered and dissolved in 20 mM acetate buffer ofpH 4.7. The solution was passed through a 10-DG column (BioRad Co.) toexchange the buffer for 20 mM acetate buffer of pH 4.7 containing 30%saturated ammonium sulfate (this solution is also referred to as “crudediglycosidase”). This solution was applied to a hydrophobicchromatography (HiLoad 16/10 Phenyl Sepharose High Performance(Pharmacia)) to separate a fraction showing diglycosidase activity fromfractions showing β-glucosidase and β-xylosidase activities. Elution wasstarted at room temperature with 20 mM acetate buffer containing 30%saturated ammonium sulfate at a flow rate of 2 mL/min and elution wascarried out by linear gradient of 30 to 0%. A fraction showingdiglycosidase activity was eluted at 10 to 12.5% saturated ammoniumsulfate concentration. The diglycosidase fraction recovered wasconcentrated and a centrifuged supernatant was charged onto 10-DG columnto exchange the solution for 25 mM tris-hydrochloride buffer of pH 7.1.This liquid was applied to an isoelectric chromatography (Mono-P HR5/20(Pharmacia)) and elution was started at room temperature with polybuffer74 of pH 5.0 at 1 mL/min. The aimed diglycosidase activity was elutedfrom pH 6.2 to pH 6.3. Since a single band was obtained on an SDSelectrophoresis of the fraction, it was proved that diglycosidase(hereinafter, also referred to as “purified diglycosidase”) could bepurified.

EXAMPLE 2 Reactivity of Diglycosidase Toward Various IsoflavoneGlycosides

[0105] Diglycosidase was diluted with an acetate buffer of pH 4.0 toprepare a 0.75 AU/mL enzyme solution. As references, a similar operationwas conducted using β-glucosidase (manufactured by Fluka) derived fromAspergillus niger, β-glucosidase (manufactured by Sigma) derived fromalmond, β-xylosidase derived from pectinase G (manufactured by AmanoEnzyme Inc.). Each of purified products of isoflavone glycosides(glycitin, acetylglycitin, malonylglycitin, daidzin, acetyldaidzin,malonyldaidzin, genistin, acetylgenistin, malonylgenistin, allmanufactured by Nacalai Tesque, Inc.) was dissolved in methanol toprepare each 2 mM substrate solution. The reaction was carried out bymixing 10 μL of a substrate solution, 200 μL of 20 mM acetate buffer (pH4.0), and 40 μL of each purified enzyme solution at a total liquidvolume of 250 μL. The reaction was carried out at 55° C. and isolationof an aglycon isoflavone from an isoflavone glycoside in the reactionmixture was detected by HLPC at 0, 1, 3, and 6 hours of the reaction.

[0106] HLPC Analysis

[0107] To the reaction mixture was added 700 μL of ethanol, followed bystirring and ultrasonication. After centrifugation at 15,000 rpm and 4°C. for 10 minutes, the supernatant was filtered through a filter andthen the filtrate was applied to HPLC.

[0108] The isoflavone glycoside and aglycon isoflavone contained in thefiltrate was separated and detected by a high performance liquidchromatography (HPLC, Shimadzu CLASS LC-10 system) using TOSOH TSK gelODS-80TM column (manufactured by Tosoh Corporation). The filtratecontaining an isoflavone glycoside and an aglycon isoflavone wasinjected into the column by means of an auto-injector (Shimadzu,SIL-10AXL) and elution was started with a solution containing 2% ofeluting solution A (acetonitrile) and 98% of eluting solution B (10%acetic acid solution), and after 5 minutes, continued by a linearconcentration gradient finishing with a solution of 50% of elutingsolution A and 50% of eluting solution B. Total flow rate was 0.8 mL/minand 12 kinds of isoflavone glycosides and aglycon isoflavones, i.e.,glycitin, daidzin, genistin, 6″-O-acetylglycitin, 6″-O-acetyldaidzin,6″-O-acetylgenistin, 6″-O-malonylglycitin, 6″-O-malonyldaidzin,6″-O-malonylgenistin, glycitein, daidzein, and genistein can beseparated. The absorbance at 260 nm was detected by a UV detector(Shimadzu, SPD-10AV). Using purified products (manufactured by NacalaiTesque, Inc.) of the above isoflavone glycosides and aglyconisoflavones, the isoflavone glycoside and aglycon isoflavone werequantitatively determined according to a calibration curve method. Bythe way, unless otherwise stated, the measuring conditions of HPLCherein mean those described in the example.

[0109] As a result, β-glucosidase derived from Aspergillus niger couldact well on 3 types of glucoside isoflavones of glycitin, daidzin, andgenistin, but the reactivity on modified glycosides was very low.β-Glucosidase derived from a plant exhibits a low efficiency ofdecomposing glycoside isoflavones and no action was observed on modifiedisoflavones. On the other hand, diglycosidase could cleave all theisoflavone glycosides at a high efficiency. Of these, a high efficiencywas observed toward acetylglucoside isoflavones. From the above results,diglycosidase was found to isolate aglycon isoflavones from isoflavoneglycosides through cleavage very efficiently (Table 1).

[0110] In addition, it was found that the efficiency of decomposingisoflavone glucosides, especially genistin could be further enhanced bythe combined use of β-glucosidase in addition to the enzyme. TABLE 1Reaction time (h) 0 1 3 6 Purified enzyme Substrate glucoside aglyconglucoside aglycon glucoside aglycon glucoside aglycon Diglycosidasederived from glycitin 100 0 2.1 97.9 0.2 99.8 0.3 99.7 P. multicolordaidzin 99.1 0.9 29.9 70.1 18.2 81.8 11.5 88.5 genistin 100 0 100 0 1000 100 0 malonylglycitin 100 0 21.7 78.3 15.5 84.5 14 86 malonyldaidzin100 0 49.2 50.8 39.9 60.1 38.5 61.5 malonylgenistin 100 0 25.8 74.2 17.982.1 17.7 82.3 acetylglycitin 100 0 2 98 2.1 97.9 2.5 97.5 acetyldaidzin100 0 10.4 89.6 3.8 96.2 3.8 96.2 acetylgenistin 100 0 0.2 99.8 0.1 99.90.2 99.8 Diglycosidase derived from glycitin 100 0 99.5 0.5 100 0 100 0A. fumigatus daidzin 99.1 0.9 92.9 7.1 85.3 14.7 75.2 24.8 genistin 1000 100 0 100 0 100 0 malonylglycitin 100 0 99.6 0.4 99.2 0.8 98.6 1.4malonyldaidzin 100 0 90 10 79.5 20.5 67.7 32.3 malonylgenistin 100 097.6 2.4 94.8 5.2 91.8 8.2 acetylglycitin 100 0 94 6 86.5 13.5 77.3 22.7acetyldaidzin 100 0 73.9 26.1 52.8 47.2 33.5 66.5 acetylgenistin 100 087.4 12.6 77.1 22.9 64.8 35.2 β-Xylosidase derived from glycitin 100 099.2 0.8 97.6 2.4 95.5 4.5 pectinase G daidzin 99.1 0.9 90.9 9.1 76 2457.9 42.1 genistin 100 0 98.5 1.5 98.2 1.8 98.2 1.8 malonylglycitin 1000 100 0 100 0 99.6 0.4 malonyldaidzin 100 0 99.6 0.4 98.4 1.6 96.7 3.3malonylgenistin 100 0 100 0 99.4 0.6 98 2 acetylglycitin 100 0 100 0 1000 100 0 acetyldaidzin 100 0 90.3 9.7 98.7 1.3 97.7 2.3 acetylgenistin100 0 100 0 99.8 0.2 99.5 0.5 β-Glucosidase derived from glycitin 100 050.5 49.5 11.7 88.3 4.5 95.5 A. niger daidzin 100 0 0 100 0 100 0 100genistin 100 0 0 100 0 100 0 100 malonylglycitin 100 0 98.8 1.2 95.6 4.490.9 9.1 malonyldaidzin 100 0 95.5 4.5 90.7 9.3 84 16 malonylgenistin100 0 96.9 3.1 93 7 87.9 12.1 acetylglycitin 100 0 100 0 99 1 98.1 1.9acetyldaidzin 100 0 95.6 4.4 93.6 6.4 91 9 acetylgenistin 100 0 98.6 1.496.7 3.3 93.8 6.2 β-Glucosidase derived from glycitin 100 0 98.4 1.698.1 1.9 98.1 1.9 almond daidzin 99.1 0.9 90.9 9.1 89.2 10.8 88.7 11.3genistin 100 0 90.6 9.4 88.6 11.4 88.2 11.8 malonylglycitin 100 0 100 0100 0 100 0 malonyldaidzin 100 0 100 0 100 0 100 0 malonylgenistin 100 0100 0 100 0 100 0 acetylglycitin 100 0 100 0 100 0 100 0 acetyldaidzin99.8 0.2 99.5 0.5 99.4 0.6 99.4 0.6 acetylgenistin 100 0 100 0 100 0 1000

EXAMPLE 3 Influence of Free Glucose on Diglycosidase Activity

[0111] Purified diglycosidase was diluted with 20 mM acetate buffer ofpH 4.0 to prepare a 0.75 AU/mL enzyme solution.

[0112] With a glucose solution was mixed 10 μL of each 2 mM substratesolution described in Example 2, and 20 mM acetate buffer of pH 4.0 wasadded thereto to be a liquid volume of 210 μL. Further, 40 μL of theenzyme solution was added and reaction was carried out at a final liquidvolume of 250 μL. Adjustment of the glucose solution to be added allowsglucose to exist in the reaction mixture in the range of 0 to 20%. Thereaction was carried out at 55° C. and the existence of isoflavoneglycosides and aglycon isoflavones was detected by HLPC at 0, 0.5, 1,and 3 hours of the reaction.

[0113] When the results were compared assuming that isolated aglyconamount at the glucose concentration of 0% and the reaction time of 0.5hour is 100% using each isoflavone glycoside as the substrate, theconverting efficiency of diglycosidase into aglycon isoflavone is hardlyinhibited by the increase of free glucose concentration. To thecontrary, increase of the converting efficiency was observed until 8%glucose concentration. Therefore, in the conversion into aglyconisoflavone by diglycosidase, no inhibition by glucose was observed up to20% concentration (Table 2).

[0114] Moreover, β-glucosidase used in the conventional convertingmethod into aglycon isoflavone is inhibited by glucose and largedecrease of the reaction efficiency was observed, but diglycosidase ofthe invention was found to be hardly inhibited by glucose. Therefore,the amount, kind, and usage of phytogenic materials, which are startingmaterials for converting into aglycon isoflavone, are restricted in thecase of the conventional glycosidase, but there is no such restrictionin the case of diglycosidase of the invention and the efficiency of theconversion into isoflavone aglycon is remarkably enhanced. TABLE 2Isolation of isoflavone aglycons by diglycosidase in presence of glucoseGlucose Concentration Isoflavone glycoside (%) glycitin daidzinacetylglycitin acetylgenistin acetyldaidzin malonylglycitinmalonylgenistin malonyldaidzin 0 100 100 100 100 100 100 100 100 2 96128.1 100.9 104.1 108.1 121.4 152 125.3 4 99.7 126 100.9 104.1 112.2127.6 164.7 132.1 8 100.1 129.1 100.9 104.1 115.8 128.6 163.5 125.4 2092.8 111.9 100.9 104.1 114.5 116.4 139.5 98.1

EXAMPLE 4 Examination of Temperature in the Conversion into IsoflavoneAglycons by Diglycosidase Using Soybean Materials

[0115] Into 400 μL of 20 mM acetate buffer of pH 4.0 was suspended 50 mgof each of various soybean materials (roasted soy flour (manufactured byFuji Shokuryo K.K.), soymilk (manufactured by Gitoh Shokuhin K.K.),defatted soybean (manufactured by Fuji Seiyu K.K.), concentrated soybeanprotein (manufactured by Fuji Seiyu K.K.)), whereby a substrate solutionwas prepared. Crude diglycosidase was diluted with 20 mM acetate bufferof pH 4.0 to be the glycosidase activity of 1.88 AU/mL. Fifty μL of theenzyme solution was mixed with the substrate solution and the whole wasreacted at 80, 65, 55, 45, 37, or 30° C. at the total volume of 500 μL.To the reaction mixture was added 700 μL of ethanol after 0, 1, 3, and 6hours of the reaction. After stirring and ultrasonication, the mixturewas subjected to centrifugal separation at 15,000 rpm and 4° C. for 10minutes. The supernatant was filtered through a filter and the existenceof isoflavone glycosides and aglycon isoflavones contained in thereaction mixture was detected by HLPC.

[0116] The decomposition efficiency from isoflavone glycosides intoaglycon isoflavones by an enzyme preparation containing diglycosidaseactivity was investigated during the treatment of 4 kinds of soybeanmaterials (roasted soy flour, soymilk, concentrated soybean protein,defatted soybean) with the enzyme, the isoflavone compounds beingseparated into three groups. Namely, the conversion efficiency wasanalyzed upon three groups of glycitin family (glycitin,malonylglycitin, acetylglycitin, glycitein), genistin family (genistin,malonylgenistin, acetylgenistin, genistein), and daidzin family(daidzin, malonyldaidzin, malonyldaidzin, acetyldaidzin, daidzein(Tables 3 to 14). TABLE 3 Decomposition efficiency of glycitin family inroasted soy flour Reaction Heat Reaction temperature treatment timeGlycoside Aglycon (° C.) of enzyme (h) glycitin malonylglycitinacetylglycitin glycitein 30 no 0 50.9 not detected 38.7 10.4 1 34.8 notdetected 33.8 31.5 3 not detected not detected 33.2 66.8 6 not detectednot detected 19.1 80.9 yes 6 51.4 not detected 37.5 11.1 45 no 0 51.3not detected 38.3 10.4 1 12.4 not detected 27.4 60.2 3 12.4 not detected20.3 67.4 6  5.9 not detected  9.1 85.0 yes 6 49.8 not detected 36.813.4 55 no 0 50.5 not detected 39.1 10.4 1 not detected not detected30.1 69.9 3 not detected not detected 12.1 87.9 6 not detected notdetected  6.0 94.0 yes 6 51.7 not detected 37.3 11.0 65 no 0 50.9 notdetected 38.7 10.4 1 not detected not detected 34.5 65.5 3 not detectednot detected 31.0 69.0 6 not detected not detected 29.0 71.0 yes 6 50.3not detected 39.3 10.4 80 no 0 51.3 not detected 38.3 10.4 1 19.2 notdetected 40.1 40.8 3 20.5 not detected 38.4 41.1 6 33.6 not detected35.7 30.7 yes 6 50.6 not detected 39.4  9.9

[0117] TABLE 4 Decomposition efficiency of genistin family in roastedsoy flour Reaction Heat Reaction temperature treatment time GlycosideAglycon (° C.) of enzyme (h) genistin malonylgenistin acetylgenistingenistein 30 no 0 49.7 not detected 45.5  4.9 1 25.7 not detected 45.329.0 3 12.0 not detected 43.8 44.2 6  5.5 not detected 40.6 53.8 yes 650.3 not detected 43.8  5.8 45 no 0 50.2 not detected 45.3  4.4 1  3.7not detected 43.6 52.7 3  5.6 not detected 38.5 55.9 6  1.2 not detected31.7 67.1 yes 6 51.7 not detected 39.5  8.8 55 no 0 49.7 not detected45.7  4.6 1  1.2 not detected 42.5 56.2 3 not detected not detected 34.066.0 6 not detected not detected 27.3 72.7 yes 6 54.0 not detected 39.9 6.2 65 no 0 49.7 not detected 45.5  4.9 1 not detected not detected43.6 56.4 3 not detected not detected 40.5 59.5 6 not detected notdetected 38.8 61.2 yes 6 50.7 not detected 44.3  5.0 80 no 0 50.2 notdetected 45.3  4.4 1  7.8 not detected 48.5 43.6 3  9.1 not detected48.2 42.7 6 18.4 not detected 47.7 33.9 yes 6 52.2 not detected 43.4 4.3

[0118] TABLE 5 Decomposition efficiency of daidzin family in roasted soyflour Reaction Heat Reaction temperature treatment time GlycosideAglycon (° C.) of enzyme (h) daidzin malonyldaidzin acetyldaidzindaidzein 35 no 0 48.6 not detected 47.8  3.6 1 11.7 not detected 46.941.4 3 not detected not detected 45.3 54.7 6 not detected not detected39.6 60.4 yes 6 50.3 not detected 43.8  5.8 45 no 0 48.6 not detected47.7  3.7 1  3.6 not detected 42.9 53.5 3  2.7 not detected 37.9 59.4 6 5.4 not detected 28.5 66.2 yes 6 49.4 not detected 42.5  8.1 55 no 048.8 not detected 47.5  3.7 1 not detected not detected 42.8 57.2 3 notdetected not detected 32.5 67.5 6 not detected not detected 26.7 73.3yes 6 51.1 not detected 43.6  5.3 65 no 0 48.6 not detected 47.8  3.6 1not detected not detected 43.7 56.3 3 not detected not detected 40.259.8 6 not detected not detected 37.8 62.2 yes 6 49.7 not detected 46.1 4.2 80 no 0 48.6 not detected 47.7  3.7 1  3.2 not detected 49.1 47.7 3 4.4 not detected 47.9 47.7 6 12.5 not detected 46.7 40.8 yes 6 51.3 notdetected 45.0  3.8

[0119] TABLE 6 Decomposition efficiency of glycitin family in soymilkReaction Heat Reaction temperature treatment time Glycoside Aglycon (°C.) of enzyme (h) glycitin malonylglycitin acetylglycitin glycitein 30no 0 43.9 48.0 not detected 8.1 1 not detected 51.0 not detected 49.0 3not detected 39.6 not detected 60.4 6 not detected 28.4 not detected71.6 yes 6 44.5 47.7 not detected 7.9 45 no 0 42.6 49.1 not detected 8.21 not detected 46.2 not detected 53.8 3 not detected 29.9 not detected70.1 6 not detected 15.3 not detected 84.7 yes 6 45.4 47.2 not detected7.4 55 no 0 47.8 44.6 not detected 7.6 1 not detected 39.1 not detected60.9 3 not detected 27.7 not detected 72.3 6 not detected 16.5 notdetected 83.5 yes 6 53.5 39.9 not detected 6.7 65 no 0 43.9 48.0 notdetected 8.1 1 not detected 42.4 not detected 57.6 3 not detected 34.3not detected 65.7 6 not detected 25.5 not detected 74.5 yes 6 56.8 36.0not detected 7.2 80 no 0 42.6 49.1 not detected 8.2 1 10.7 43.6 notdetected 45.7 3 22.5 32.6 not detected 44.9 6 44.2 21.3 not detected34.4 yes 6 71.4 19.3 not detected 9.3

[0120] TABLE 7 Decomposition efficiency of genistin family in soymilkReaction Heat Reaction temperature treatment time Glycoside Aglycon (°C.) of enzyme (h) genistin malonylgenistin acetylgenistin genistein 30no 0 30.7 58.5 0.9 10.0 1 not detected 56.5 not detected 43.5 3 notdetected 47.9 not detected 52.1 6 not detected 36.1 not detected 63.9yes 6 31.2 57.7 0.9 10.1 45 no 0 31.0 57.8 0.9 10.3 1 not detected 49.4not detected 50.6 3 not detected 34.7 not detected 65.3 6 not detected20.7 not detected 79.3 yes 6 34.4 57.2 0.6  7.9 55 no 0 31.6 59.1 0.6 8.7 1 not detected 46.9 not detected 53.1 3 not detected 32.5 notdetected 67.5 6 not detected 20.4 not detected 79.6 yes 6 37.4 53.3 0.7 8.6 65 no 0 30.7 58.5 0.9 10.0 1 not detected 48.9 not detected 51.1 3not detected 40.6 not detected 59.4 6 not detected 31.8 not detected68.2 yes 6 44.4 44.6 1.1  9.9 80 no 0 31.0 57.8 0.9 10.3 1 10.6 50.4 0.938.0 3 24.3 37.7 1.0 37.0 6 43.9 24.1 2.3 29.6 yes 6 64.5 22.9 2.5 10.1

[0121] TABLE 8 Decomposition efficiency of daidzin family in soymilkReaction Heat Reaction temperature treatment time Glycoside Aglycon (°C.) of enzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein 30 no 034.1 56.8 not detected 9.1 1 not detected 58.2 not detected 41.8 3 notdetected 52.8 not detected 47.2 6 not detected 46.2 not detected 53.8yes 6 34.8 56.1 not detected 9.1 45 no 0 33.9 57.2 not detected 8.9 1 6.8 50.2 not detected 43.0 3  6.9 41.1 not detected 52.0 6  6.9 30.2not detected 62.8 yes 6 36.0 55.8 not detected 8.2 55 no 0 34.2 57.3 notdetected 8.6 1 not detected 50.9 not detected 49.1 3 not detected 41.1not detected 58.9 6 not detected 31.1 not detected 68.9 yes 6 40.7 50.8not detected 8.6 65 no 0 34.1 56.8 not detected 9.1 1 not detected 51.7not detected 48.3 3 not detected 43.9 not detected 56.1 6 not detected35.3 not detected 64.7 yes 6 48.7 42.4 not detected 8.8 80 no 0 33.957.2 not detected 8.9 1  8.9 50.8 not detected 40.3 3 23.9 36.4 notdetected 39.7 6 42.9 23.0 not detected 34.1 yes 6 69.5 21.8 not detected8.8

[0122] TABLE 9 Decomposition efficiency of glycitin family inconcentrated soybean protein Reaction Heat Reaction temperaturetreatment time Glycoside Aglycon (° C.) of enzyme (h) glycitinmalonylglycitin acetylglycitin glycitein 30 no 0 52.2 0.4 36.0 11.4 147.1 0.5 35.0 17.5 3 39.1 0.5 33.4 26.9 6 28.4 0.5 30.8 40.3 yes 6 52.80.4 35.4 11.3 45 no 0 52.3 0.4 35.9 11.4 1 30.2 0.6 34.1 35.2 3 18.8 0.631.0 49.6 6  7.5 0.6 27.1 64.9 yes 6 53.2 0.5 35.0 11.3 55 no 0 52.3 0.436.0 11.2 1 14.2 0.5 33.9 51.4 3 not detected 0.5 29.8 69.7 6 notdetected 0.5 26.8 72.7 yes 6 52.9 0.4 35.3 11.3 65 no 0 52.2 0.4 36.011.4 1  5.2 0.5 35.7 58.6 3 not detected 0.5 35.5 64.0 6 not detected0.4 35.8 63.8 yes 6 52.5 0.4 35.7 11.4 80 no 0 52.3 0.4 35.9 11.4 1 39.70.4 36.0 23.8 3 40.5 0.4 35.7 23.4 6 41.7 0.3 35.2 22.8 yes 6 53.6 0.334.8 11.4

[0123] TABLE 10 Decomposition efficiency of genistin family inconcentrated soybean protein Reaction Heat Reaction temperaturetreatment time Glycoside Aglycon (° C.) of enzyme (h) genistinmalonylgenistin acetylgenistin genistein 30 no 0 49.8 not detected 43.17.1 1 41.1 not detected 43.2 15.7 3 30.3 not detected 43.0 26.7 6 19.7not detected 42.5 37.8 yes 6 51.8 not detected 41.5 6.7 45 no 0 49.7 notdetected 43.0 7.2 1 22.2 not detected 42.8 35.0 3 15.6 not detected 41.043.4 6  5.3 not detected 39.6 55.1 yes 6 54.5 not detected 38.9 6.5 55no 0 50.1 not detected 42.9 7.0 1  7.0 not detected 43.1 49.8 3  0.8 notdetected 41.1 58.1 6 not detected not detected 39.0 61.0 yes 6 52.3 notdetected 41.1 6.6 65 no 0 49.8 not detected 43.1 7.1 1 not detected notdetected 45.2 54.8 3 not detected not detected 44.0 56.0 6 not detectednot detected 43.4 56.6 yes 6 50.8 not detected 42.3 6.9 80 no 0 49.7 notdetected 43.0 7.2 1 29.6 not detected 44.0 26.4 3 30.3 not detected 43.626.1 6 32.3 not detected 43.4 24.3 yes 6 51.9 not detected 41.4 6.7

[0124] TABLE 11 Decomposition efficiency of daidzin family inconcentrated soybean protein Reaction Heat Reaction temperaturetreatment time Glycoside Aglycon (° C.) of enzyme (h) daidzinmalonyldaidzin acetyldaidzin daidzein 30 no 0 52.3 not detected 44.6 3.01 29.5 not detected 44.4 26.2 3 13.4 not detected 43.9 42.7 6  4.0 notdetected 43.5 52.5 yes 6 54.5 not detected 42.3 3.2 45 no 0 52.4 notdetected 44.5 3.1 1 10.6 not detected 44.1 45.3 3  9.6 not detected 41.548.8 6  1.7 not detected 42.9 55.5 yes 6 94.3 not detected  0.0 5.7 55no 0 52.5 not detected 44.5 3.0 1  1.0 not detected 44.0 55.0 3 notdetected not detected 40.6 59.4 6 not detected not detected 37.7 62.3yes 6 54.6 not detected 42.3 3.2 65 no 0 52.3 not detected 44.6 3.0 1 1.8 not detected 44.3 53.9 3  1.7 not detected 42.9 55.4 6  1.7 notdetected 41.6 56.7 yes 6 53.2 not detected 43.6 3.2 80 no 0 52.4 notdetected 44.5 3.1 1 22.6 not detected 45.5 31.9 3 23.3 not detected 44.732.0 6 25.5 not detected 43.8 30.7 yes 6 54.5 not detected 42.3 3.2

[0125] TABLE 12 Decomposition efficiency of glycitin family in defattedsoybean Reaction Heat Reaction temperature treatment time GlycosideAglycon (° C.) of enzyme (h) glycitin malonylglycitin acetylglycitinglycitein 30 no 0 not detected 61.3 not detected 38.7 1 not detected48.3 not detected 51.7 3 not detected 44.6 not detected 55.4 6 notdetected 45.6 not detected 54.4 yes 6 not detected 45.1 not detected54.9 45 no 0 not detected 65.2 not detected 34.8 1 not detected 44.3 notdetected 55.7 3 not detected 44.8 not detected 55.2 6 not detected 41.0not detected 59.0 yes 6 not detected 44.2 not detected 55.8 55 no 0 notdetected 61.3 not detected 38.7 1 not detected 44.8 not detected 55.2 3not detected 42.6 not detected 57.4 6 not detected 42.7 not detected57.3 yes 6 not detected 45.5 not detected 54.5 65 no 0 not detected 65.2not detected 34.8 1 not detected 45.3 not detected 54.7 3 not detected42.1 not detected 57.9 6 not detected 40.0 not detected 60.0 yes 6 notdetected 48.8 not detected 51.2 80 no 0 not detected 65.2 not detected34.8 1 not detected 46.1 not detected 53.9 3 not detected 45.0 notdetected 55.0 6 not detected 40.3 not detected 59.7 yes 6 not detected48.1 not detected 51.9

[0126] TABLE 13 Decomposition efficiency of genistin family in defattedsoybean Reaction Heat Reaction temperature treatment time GlycosideAglycon (° C.) of enzyme (h) genistin malonylgenistin acetylgenistingenistein 30 no 0 38.0 47.0 1.7 13.3 1 not detected 51.3 1.3 47.4 3 notdetected 49.1 0.7 50.2 6  7.6 45.6 0.1 46.6 yes 6 not detected 49.5 0.450.2 45 no 0 37.0 47.0 1.5 14.5 1  0.9 49.0 0.8 49.2 3  1.0 46.8 0.351.8 6  1.0 44.2 0.2 54.6 yes 6  1.3 48.1 0.2 50.4 55 no 0 38.0 47.0 1.713.3 1  0.0 48.8 0.7 50.5 3  0.0 46.1 0.2 53.7 6  0.0 43.4 0.1 56.5 yes6  0.0 47.8 0.4 51.9 65 no 0 37.0 47.0 1.5 14.5 1  0.0 48.9 1.2 49.9 3 1.8 45.2 1.2 51.8 6  3.3 41.1 1.1 54.5 yes 6 28.2 39.7 1.3 30.7 80 no 037.0 47.0 1.5 14.5 1  9.6 43.8 1.8 44.9 3 22.1 32.5 2.1 43.3 6 35.3 20.92.5 41.3 yes 6 57.8 20.1 2.4 19.6

[0127] TABLE 14 Decomposition efficiency of daidzin family in defattedsoybean Reaction Heat Reaction temperature treatment time GlycosideAglycon (° C.) of enzyme (h) daidzin malonyldaidzin acetyldaidzindaidzein 30 no 0 42.2 43.3 not detected 14.5 1 not detected 46.6 notdetected 53.4 3 not detected 45.2 not detected 54.8 6 not detected 45.1not detected 54.9 yes 6 not detected 45.4 not detected 54.6 45 no 0 40.543.3 not detected 16.2 1 not detected 45.5 not detected 54.5 3 notdetected 43.7 not detected 56.3 6 not detected 41.7 not detected 58.3yes 6 not detected 44.3 not detected 55.7 55 no 0 42.2 43.3 not detected14.5 1 not detected 45.0 not detected 55.0 3 not detected 42.7 notdetected 57.3 6 not detected 40.3 not detected 59.7 yes 6 not detected44.1 not detected 55.9 65 no 0 40.5 43.3 not detected 16.2 1 notdetected 44.7 not detected 55.3 3 not detected 41.6 not detected 58.4 6not detected 37.3 not detected 62.7 yes 6 32.8 35.2 not detected 32.0 80no 0 40.5 43.3 not detected 16.2 1  7.8 39.8 not detected 52.4 3 19.228.8 not detected 52.0 6 30.4 17.8 not detected 51.7 yes 6 60.9 17.4 notdetected 21.7

[0128] All three groups of isoflavone glucosides were decomposed at allthe temperature ranges tested, and promptly at 37 to 65° C.,particularly 55° C. Furthermore, in defatted soybean, endogenousβ-glucosidase also participates in the decomposition. It is revealedthat the decomposition of modified glucoside glycosides, most of whichis considered to occur by the action of diglycosidase, easily occurs ata temperature of 37 to 55° C. Among three groups of aglycon isoflavones,maximum isolation of aglycons was observed in the reaction at 55° C. for6 hours. In the case of soy flour, glycitein was about 94%, genisteinabout 74%, and daidzein about 73%. In the case of soymilk, glycitein wasabout 84%, genistein about 80%, and daidzein about 70%. In the case ofconcentrated soybean protein, glycitein was about 73%, genistein about61%, and daidzein about 62%. In the case of defatted soybean, glyciteinwas about 57%, genistein about 57%, and daidzein about 60%.

[0129] From these results, it was revealed that the decomposition ofmodified glucoside glycosides by diglycosidase efficiently occurred at atemperature of 37 to 65° C., particularly around 55° C.

EXAMPLE 5 Examination of pH in the Conversion into Isoflavone Aglyconsby Diglycosidase Using Soybean Materials

[0130] A substrate solution of 450 μL was prepared by suspending 50 mgof each soybean material (roasted soy flour (manufactured by FujiShokuryo K.K.), soymilk (manufactured by Gitoh Shokuhin K.K.), defattedsoybean (manufactured by Fuji Seiyu K.K.), concentrated soybean protein(manufactured by Fuji Seiyu K.K.)), and adjusting the pH to 2 to 11 withhydrochloric acid or sodium hydroxide. Each enzyme solution of pH 2-11wherein diglycosidase activity of crude diglycosidase was adjusted to1.88 AU/mL was added thereto in an amount of 50 μL and the whole wasreacted at 55° C. at the total volume of 500 μL. To the reaction mixturewas added 700 μL of ethanol after 0, 1, 3, and 6 hours of the reaction.After stirring and ultrasonication, the mixture was subjected tocentrifugal separation at 15,000 rpm and 4° C. for 10 minutes. Thesupernatant was filtered through a filter and the existence ofisoflavone glycosides and aglycon isoflavones contained in the reactionmixture was detected by HLPC (Tables 15 to 26). TABLE 15 Decompositionefficiency of glycitin family in roasted soy flour Heat Reactiontreatment time Glycoside Aglycon Reaction pH of enzyme (h) glycitinmalonylglycitin acetylglycitin glycitein pH2 no 0 56.8 not detected 35.37.9 1 56.6 not detected 34.0 9.4 3 56.3 not detected 34.2 9.5 6 54.4 notdetected 35.5 10.1 yes 6 54.4 not detected 35.2 10.4 pH3 no 0 56.8 notdetected 35.3 7.9 1 not detected not detected 38.7 61.3 3 not detectednot detected 30.8 69.2 6 not detected not detected 31.7 68.3 yes 6 55.1not detected 35.6 9.3 pH4 no 0 56.6 not detected 34.8 8.6 1 not detectednot detected 18.8 81.2 3 not detected not detected  8.2 91.8 6 notdetected not detected not detected 100.0 yes 6 55.1 not detected 35.99.0 pH5 no 0 56.6 not detected 34.8 8.6 1 not detected not detected 22.977.1 3 not detected not detected  6.5 93.5 6 not detected not detectednot detected 100.0 yes 6 55.4 not detected 35.2 9.4 pH6.5 no 0 55.9 notdetected 35.4 8.8 1 not detected not detected 35.0 65.0 3 not detectednot detected 19.7 80.3 6 not detected not detected 11.9 88.1 yes 6 54.8not detected 35.4 9.8 pH8.5 no 0 59.4 not detected 31.0 9.6 1 39.4 notdetected 32.4 28.2 3 29.3 not detected 30.6 40.1 6 22.4 not detected30.2 47.4 yes 6 59.2 not detected 31.7 9.1

[0131] TABLE 16 Decomposition efficiency of genistin family in roastedsoy flour Heat Reaction treatment time Glycoside Aglycon Reaction pH ofenzyme (h) genistin malonylgenistin acetylgenistin genistein pH2 no 050.0 not detected 45.2 4.8 1 49.7 not detected 45.0 5.3 3 50.7 notdetected 43.1 6.2 6 52.7 not detected 40.7 6.5 yes 6 52.8 not detected41.2 6.0 pH3 no 0 50.0 not detected 45.2 4.8 1  3.4 not detected 45.251.4 3 not detected not detected 41.9 58.1 6 not detected not detected43.5 56.5 yes 6 49.9 not detected 45.1 5.0 pH4 no 0 49.9 not detected45.4 4.7 1 not detected not detected 36.3 63.7 3 not detected notdetected 23.4 76.6 6 not detected not detected 13.4 86.6 yes 6 50.2 notdetected 45.4 4.4 pH5 no 0 49.9 not detected 45.4 4.7 1 not detected notdetected 39.9 60.1 3 not detected not detected 26.9 73.1 6 not detectednot detected 17.6 82.4 yes 6 52.6 not detected 42.0 5.3 pH6.5 no 0 49.4not detected 45.8 4.8 1 not detected not detected 45.3 54.7 3 notdetected not detected 38.8 61.2 6 not detected not detected 34.0 66.0yes 6 54.8 not detected 39.2 6.0 pH8.5 no 0 55.8 not detected 39.5 4.6 128.7 not detected 39.5 31.8 3 19.2 not detected 39.2 41.6 6 14.5 notdetected 37.3 48.2 yes 6 59.5 not detected 35.2 5.3

[0132] TABLE 17 Decomposition efficiency of daidzin family in roastedsoy flour Heat Reaction treatment time Glycoside Aglycon Reaction pH ofenzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein pH2 no 0 49.1not detected 47.4 3.5 1 47.6 not detected 46.9 5.4 3 50.3 not detected45.0 4.8 6 53.1 not detected 42.4 4.5 yes 6 53.8 not detected 42.4 3.8pH3 no 0 49.1 not detected 47.4 3.5 1 not detected not detected 46.054.0 3 not detected not detected 39.6 60.4 6 not detected not detected40.7 59.3 yes 6 49.0 not detected 47.4 3.7 pH4 no 0 48.7 not detected47.7 3.5 1 not detected not detected 34.4 65.6 3 not detected notdetected 20.5 79.5 6 not detected not detected 11.1 88.9 yes 6 48.6 notdetected 47.9 3.5 pH5 no 0 48.7 not detected 47.7 3.5 1 not detected notdetected 38.5 61.5 3 not detected not detected 24.7 75.3 6 not detectednot detected 15.2 84.8 yes 6 50.6 not detected 45.0 4.4 pH6.5 no 0 48.4not detected 48.2 3.4 1 not detected not detected 45.4 54.6 3 notdetected not detected 39.2 60.8 6 not detected not detected 34.4 65.6yes 6 51.9 not detected 43.4 4.7 pH8.5 no 0 54.1 not detected 42.1 3.8 120.4 not detected 42.7 36.9 3 13.3 not detected 40.3 46.4 6 10.2 notdetected 38.0 51.8 yes 6 58.8 not detected 37.3 3.9

[0133] TABLE 18 Decomposition efficiency of glycitin family in soymilkHeat Reaction treatment time Glycoside Aglycon Reaction pH of enzyme (h)glycitin malonylglycitin acetylglycitin glycitein pH2.3 no 0 50.2 42.6not detected 7.2 1 not detected 51.3 not detected 48.7 3 not detected47.7 not detected 52.3 6 not detected 46.5 not detected 53.5 yes 6 51.741.3 not detected 6.9 pH3.5 no 0 50.2 42.6 not detected 7.2 1 notdetected 21.9 not detected 78.1 3 not detected not detected not detected100.0 6 not detected not detected not detected 100.0 yes 6 54.2 38.2 notdetected 7.6 pH4.8 no 0 50.7 43.2 not detected 6.2 1 not detected 37.0not detected 63.0 3 not detected 21.1 not detected 78.9 6 not detected 8.3 not detected 91.7 yes 6 54.3 39.2 not detected 6.5 pH6.2 no 0 50.743.2 not detected 6.2 1 not detected 46.4 not detected 53.6 3 notdetected 34.5 not detected 65.5 6 not detected 29.6 not detected 70.4yes 6 54.2 37.6 not detected 8.2 pH7.2 no 0 50.0 43.9 not detected 6.1 1not detected 48.8 not detected 51.2 3 not detected 46.2 not detected53.8 6 not detected 41.6 not detected 58.4 yes 6 56.9 37.5 not detected5.6 pH11.6 no 0 54.3 39.4 not detected 6.2 1 not detected 50.4 notdetected 49.6 3 not detected 46.5 not detected 53.5 6 not detected 44.0not detected 56.0 yes 6 59.7 34.4 not detected 5.8

[0134] TABLE 19 Decomposition efficiency of genistin family in soymilkHeat Reaction treatment time Glycoside Aglycon Reaction pH of enzyme (h)genistin malonylgenistin acetylgenistin genistein pH2.3 no 0 31.5 58.20.6 9.8 1 not detected 57.5 0.7 41.8 3 not detected 54.1 0.8 45.1 6 notdetected 52.1 0.6 47.3 yes 6 35.5 54.3 0.6 9.6 pH3.5 no 0 31.5 58.2 0.69.8 1 not detected 27.9 not detected 72.1 3 not detected 7.5 notdetected 92.5 6 not detected 2.7 not detected 97.3 yes 6 37.0 52.7 0.59.8 pH4.8 no 0 31.7 58.6 0.5 9.1 1 not detected 41.0 not detected 59.0 3not detected 23.9 not detected 76.1 6 not detected 11.3 not detected88.7 yes 6 37.8 51.9 0.6 9.7 pH6.2 no 0 31.7 58.6 0.5 9.1 1 not detected52.6 not detected 47.4 3 not detected 41.2 not detected 58.8 6 notdetected 35.8 not detected 64.2 yes 6 38.3 51.5 0.6 9.5 pH7.2 no 0 31.257.0 0.7 11.1 1 not detected 56.9 not detected 43.1 3 not detected 51.5not detected 48.5 6 not detected 46.4 not detected 53.6 yes 6 38.9 51.10.7 9.3 pH11.6 no 0 34.8 54.2 0.5 10.5 1 not detected 57.7 not detected42.3 3 not detected 52.6 not detected 47.4 6 not detected 49.5 notdetected 50.5 yes 6 41.3 47.7 0.9 10.1

[0135] TABLE 20 Decomposition efficiency of daidzin family in soymilkHeat Reaction treatment time Glycoside Aglycon Reaction pH of enzyme (h)daidzin malonyldaidzin acetyldaidzin daidzein pH2.3 no 0 34.4 55.1 notdetected 10.5 1 not detected 54.7 not detected 45.3 3 not detected 51.1not detected 48.9 6 not detected 48.5 not detected 51.5 yes 6 38.8 51.0not detected 10.2 pH3.5 no 0 34.4 55.1 not detected 10.5 1 not detected35.6 not detected 64.4 3 not detected 15.1 not detected 84.9 6 notdetected  7.4 not detected 92.6 yes 6 40.4 49.1 not detected 10.6 pH4.8no 0 34.9 56.2 not detected 9.0 1 not detected 47.4 not detected 52.6 3not detected 33.9 not detected 66.1 6 not detected 20.3 not detected79.7 yes 6 41.2 49.7 not detected 9.1 pH6.2 no 0 34.9 56.2 not detected9.0 1 not detected 54.1 not detected 45.9 3 not detected 46.4 notdetected 53.6 6 not detected 41.9 not detected 58.1 yes 6 40.6 49.7 notdetected 9.7 pH7.2 no 0 34.9 56.1 not detected 9.0 1 not detected 56.4not detected 43.6 3 not detected 52.4 not detected 47.6 6 not detected48.2 not detected 51.8 yes 6 41.5 48.3 not detected 10.2 pH11.6 no 038.8 52.5 not detected 8.7 1 not detected 56.4 not detected 43.6 3 notdetected 50.4 not detected 49.6 6 not detected 47.7 not detected 52.3yes 6 44.6 46.1 not detected 9.3

[0136] TABLE 21 Decomposition efficiency of glycitin family inconcentrated soybean protein Heat treatment Reaction Reaction of timeGlycoside Aglycon pH enzyme (h) glycitin malonylglycitin acetylglycitinglycitein pH1.6 no 0 52.3 0.4 35.9 11.3 1 52.9 0.4 35.1 11.6 3 54.3 0.433.5 11.7 6 56.1 0.4 31.8 11.7 yes 6 57.9 0.4 30.9 10.8 pH2.7 no 0 52.30.4 35.9 11.3 1 38.0 0.5 36.6 25.0 3 37.2 0.5 36.5 25.7 6 33.7 0.5 36.629.2 yes 6 52.7 0.4 35.4 11.5 pH3.7 no 0 52.4 0.4 36.1 11.1 1 notdetected 0.5 32.9 66.7 3 not detected 0.4 26.1 73.5 6 not detected 0.422.1 77.5 yes 6 52.4 0.4 35.9 11.3 pH5.1 no 0 52.4 0.4 36.1 11.1 1 4.10.6 33.2 62.1 3 not detected 0.6 26.0 73.4 6 not detected 0.6 21.0 78.5yes 6 52.3 0.4 35.8 11.5 pH6.6 no 0 52.2 0.4 36.1 11.3 1 22.6 0.4 34.842.2 3 6.9 0.4 32.4 60.3 6 2.0 0.4 29.5 68.1 yes 6 53.3 0.4 35.2 11.1pH8.6 no 0 54.9 0.4 33.0 11.7 1 51.6 0.4 32.6 15.4 3 45.9 0.4 32.6 21.06 36.7 0.4 32.0 30.9 yes 6 56.8 0.3 31.6 11.3

[0137] TABLE 22 Decomposition efficiency of genistin family inconcentrated soybean protein Heat treatment Reaction Reaction of timeGlycoside Aglycon pH enzyme (h) genistin malonylgenistin acetylgenistingenistein pH1.6 no 0 49.7 not detected 42.5 7.8 1 50.2 not detected 42.27.6 3 51.6 not detected 40.6 7.8 6 53.3 not detected 38.9 7.9 yes 6 54.4not detected 37.7 7.9 pH2.7 no 0 49.7 not detected 42.5 7.8 1 23.1 notdetected 44.3 32.6 3 22.1 not detected 43.7 34.2 6 17.5 not detected44.0 38.4 yes 6 50.5 not detected 42.5 7.0 pH3.7 no 0 50.7 not detected43.1 6.2 1 not detected not detected 43.5 56.5 3 not detected notdetected 36.9 63.1 6 not detected not detected 30.7 69.3 yes 6 50.6 notdetected 43.0 6.4 pH5.1 no 0 50.7 not detected 43.1 6.2 1 not detectednot detected 45.2 54.8 3 not detected not detected 40.2 59.8 6 notdetected not detected 35.6 64.4 yes 6 51.4 not detected 42.4 6.2 pH6.6no 0 50.4 not detected 43.0 6.5 1 13.9 not detected 43.6 42.4 3 3.8 notdetected 42.8 53.3 6 1.7 not detected 41.0 57.2 yes 6 53.2 not detected40.6 6.2 pH8.6 no 0 53.8 not detected 39.7 6.5 1 50.4 not detected 38.810.8 3 45.1 not detected 39.0 15.8 6 36.3 not detected 38.5 25.2 yes 656.2 not detected 37.6 6.2

[0138] TABLE 23 Decomposition efficiency of daidzin family inconcentrated soybean protein Heat treatment Reaction Reaction of timeGlycoside Aglycon pH enzyme (h) daidzin malonyldaidzin acetyldaidzindaidzein pH1.6 no 0 52.5 not detected 44.5 3.0 1 53.0 not detected 43.73.3 3 55.0 not detected 41.7 3.3 6 57.2 not detected 39.4 3.4 yes 6 59.0not detected 38.1 2.9 pH2.7 no 0 52.5 not detected 44.5 3.0 1 12.9 notdetected 46.4 40.7 3 12.5 not detected 46.0 41.6 6 9.3 not detected 45.845.0 yes 6 53.0 not detected 43.9 3.2 pH3.7 no 0 52.5 not detected 44.63.0 1 not detected not detected 41.6 58.4 3 not detected not detected33.0 67.0 6 not detected not detected 25.2 74.8 yes 6 52.5 not detected44.4 3.1 pH5.1 no 0 52.5 not detected 44.6 3.0 1 not detected notdetected 43.4 56.6 3 not detected not detected 37.0 63.0 6 not detectednot detected 31.2 68.8 yes 6 53.4 not detected 43.4 3.2 pH6.6 no 0 52.1not detected 44.8 3.0 1 5.0 not detected 43.9 51.0 3 not detected notdetected 42.9 57.1 6 not detected not detected 41.2 58.8 yes 6 55.1 notdetected 41.8 3.2 pH8.6 no 0 56.1 not detected 40.7 3.2 1 50.8 notdetected 39.2 9.9 3 45.0 not detected 39.0 15.9 6 34.5 not detected 38.127.4 yes 6 59.1 not detected 37.7 3.2

[0139] TABLE 24 Decomposition efficiency of glycitin family in defattedsoybean Heat treatment Reaction Reaction of time Glycoside Aglycon pHenzyme (h) glycitin malonylglycitin acetylglycitin glycitein pH2.6 no 0not detected 100.0 not detected not detected 1 not detected 100.0 notdetected not detected 3 not detected 100.0 not detected not detected 6not detected 100.0 not detected not detected yes 6 not detected 100.0not detected not detected pH3.4 no 0 not detected 100.0 not detected notdetected 1 not detected 100.0 not detected not detected 3 not detected100.0 not detected not detected 6 not detected 100.0 not detected notdetected yes 6 not detected 100.0 not detected not detected pH4.8 no 0not detected 64.0 not detected 36.0 1 not detected 40.1 not detected59.9 3 not detected 37.5 not detected 62.5 6 not detected 31.9 notdetected 68.1 yes 6 not detected 56.2 not detected 43.8 pH5.4 no 0 notdetected 64.0 not detected 36.0 1 not detected 45.1 not detected 54.9 3not detected 100.0 not detected not detected 6 not detected 32.3 notdetected 67.7 yes 6 not detected 39.3 not detected 60.7 pH6.6 no 0 notdetected 39.6 not detected 60.4 1 not detected 52.9 not detected 47.1 3not detected 53.6 not detected 46.4 6 not detected 58.1 not detected41.9 yes 6 not detected 50.3 not detected 49.7 pH7.8 no 0 not detected66.8 not detected 33.2 1 not detected 57.5 not detected 42.5 3 notdetected 53.2 not detected 46.8 6 not detected 49.1 not detected 50.9yes 6 not detected 55.8 not detected 44.2

[0140] TABLE 25 Decomposition efficiency of genistin family in defattedsoybean Heat treatment Reaction Reaction of time Glycoside Aglycon pHenzyme (h) genistin malonylgenistin acetylgenistin genistein pH2.6 no 042.0 43.8 1.8 12.4 1 36.3 44.4 1.7 17.7 3 20.3 45.4 1.7 32.7 6 20.1 43.51.9 34.6 yes 6 44.3 42.0 1.6 12.1 pH3.4 no 0 42.0 43.8 1.8 12.4 1 notdetected 41.6 1.5 56.9 3 not detected 36.4 1.1 62.5 6 not detected 30.40.8 68.8 yes 6 44.3 39.8 1.8 14.1 pH4.8 no 0 40.5 43.2 1.8 14.5 1 notdetected 42.8 1.0 56.1 3 not detected 37.5 0.6 61.9 6 not detected 31.10.4 68.5 yes 6 31.8 41.7 1.3 25.2 pH5.4 no 0 40.5 43.2 1.8 14.5 1 notdetected 48.0 0.5 51.5 3 not detected 44.6 not detected 55.4 6 notdetected 39.7 not detected 60.3 yes 6 6.2 45.8 not detected 48.0 pH6.6no 0 39.2 47.1 1.7 12.0 1 not detected 50.1 0.6 49.3 3 not detected 48.30.3 51.4 6 not detected 46.1 0.2 53.7 yes 6 7.7 45.7 0.2 46.4 pH7.8 no 040.4 45.8 1.6 12.2 1 15.7 46.6 1.0 36.7 3 12.3 44.4 0.8 42.5 6 11.4 42.20.7 45.7 yes 6 37.5 39.9 0.6 22.0

[0141] TABLE 26 Decomposition efficiency of daidzin family in defattedsoybean Heat treatment Reaction Reaction of time Glycoside Aglycon pHenzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein pH2.6 no 0 45.841.0 not detected 13.2 1 37.5 41.2 not detected 21.3 3 17.3 41.8 notdetected 40.9 6 16.9 40.2 not detected 42.9 yes 6 48.0 38.9 not detected13.0 pH3.4 no 0 45.8 41.0 not detected 13.2 1 not detected 39.4 notdetected 60.6 3 not detected 34.5 not detected 65.5 6 not detected 29.0not detected 71.0 yes 6 47.8 37.1 not detected 15.1 pH4.8 no 0 43.5 40.7not detected 15.7 1 not detected 40.5 not detected 59.5 3 not detected36.7 not detected 63.3 6 not detected 30.5 not detected 69.5 yes 6 37.138.4 not detected 24.5 pH5.4 no 0 43.5 40.7 not detected 15.7 1 notdetected 44.2 not detected 55.8 3 not detected 41.5 not detected 58.5 6not detected 36.9 not detected 63.1 yes 6 not detected 44.9 not detected55.1 pH6.6 no 0 43.6 43.1 not detected 13.2 1 not detected 45.9 notdetected 54.1 3 not detected 44.0 not detected 56.0 6 not detected 41.8not detected 58.2 yes 6 9.3 41.2 not detected 49.5 pH7.8 no 0 44.7 42.0not detected 13.3 1 13.3 43.6 not detected 43.1 3 10.1 41.4 not detected48.4 6 9.4 38.7 not detected 51.9 yes 6 41.6 36.7 not detected 21.7

[0142] From these results, maximum pH was found to be in the range of3.5 to 5. Specifically, in the case of roasted soy flour, after thereaction at pH 4 for 6 hours, the existing ratio of aglycon of eachisoflavone family was as follows: glycitein 100%, genistein 87%, anddaidzein 89%. In the case of soymilk, almost complete decomposition ofisoflavone glycosides occurred after the reaction at pH 3.5 for 6 hours,and glycitein was 100%, genistein 97%, and daidzein 93%. In the case ofconcentrated soybean protein, after the reaction at pH 3.7 for 6 hours,glycitein was 78%, genistein 69%, and daidzein 75%. In the case ofdefatted soybean, maximum isolation of aglycons was observed after thereaction at pH 3.4 for 6 hours, and glycitein was 68%, genistein 69%,and daidzein 71%.

EXAMPLE 6 Examination of Substrate Concentration in the Conversion intoIsoflavone Aglycons by Diglycosidase Using Soybean Materials

[0143] Into 20 mM acetate buffer of pH 4.0 was suspended 0.1 g, 0.25 g,0.5 g, 1.0 g, or 1.5 g of each of various soybean materials (roasted soyflour (manufactured by Fuji Shokuryo K.K.), soymilk (manufactured byGitoh Shokuhin K.K.), defatted soybean (manufactured by Fuji SeiyuK.K.), concentrated soybean protein (manufactured by Fuji Seiyu K.K.)).The pH of the suspension was measured and the liquid volume was adjustedto 4.5 mL while the pH was adjusted to 4.0 with 1N hydrochloric acid. Anenzyme solution wherein diglycosidase activity of crude diglycosidasewas adjusted to 1.88 AU/mL was added thereto in an amount of 0.5 mL,whereby the final liquid volume was 5.0 mL. Namely, the ratio of thesoybean material in the reaction mixture was 2%, 5%, 10%, 20%, or 30%(w/v). The whole was reacted at 55° C. under shaking. To 5 mL of thereaction mixture was added 7 mL of ethanol after 0, 1, 3, and 6 hours ofthe reaction. After ultrasonication, the mixture was thoroughly mixed.It was subjected to centrifugal separation at 2,000 rpm and roomtemperature for 5 minutes. Then, 1 μL of the supernatant was placed in a1.5 mL microtube and was subjected to centrifugal separation at 15,000rpm and 4° C. for 10 minutes. The supernatant was filtered through afilter and each sample was suitably diluted by a factor of 1 to 6depending on the substrate concentration. Fifty μL of the dilutedsolution was analyzed by HLPC (Tables 27 to 38). TABLE 27 Decompositionefficiency of glycitin family in roasted soy flour Substrate HeatReaction concentration treatment time Glycoside Aglycon (%) of enzyme(h) glycitin malonylglycitin acetylglycitin glycitein 2 no 0 50.2 notdetected 33.6 16.2 1 not detected not detected not detected 100.0 3 notdetected not detected not detected 100.0 6 not detected not detected notdetected 100.0 yes 6 53.4 not detected 34.4 12.2 5 no 0 54.8 notdetected 33.8 11.4 1 not detected not detected 11.8 88.2 3 not detectednot detected 7.2 92.8 6 not detected not detected not detected 100.0 yes6 54.8 not detected 33.8 11.4 10 no 0 70.3 not detected 24.2 5.5 1 notdetected not detected 16.7 83.3 3 not detected not detected 9.7 90.3 6not detected not detected not detected 100.0 yes 6 55.0 not detected35.1 9.9 20 no 0 59.6 not detected 31.8 8.6 1 not detected not detected22.9 77.1 3 not detected not detected 13.8 86.2 6 not detected notdetected 10.0 90.0 yes 6 56.0 not detected 35.0 9.1 30 no 0 55.9 notdetected 35.3 8.8 1 not detected not detected 28.4 71.6 3 not detectednot detected 24.4 75.6 6 not detected not detected 13.7 86.3 yes 6 56.9not detected 34.4 8.7

[0144] TABLE 28 Decomposition efficiency of genistin family in roastedsoy flour Substrate Heat Reaction concentration treatment time GlycosideAglycon (%) of enzyme (h) genistin malonylgenistin acetylgenistingenistein 2 no 0 33.2 not detected 44.7 22.1 1 not detected not detected10.6 89.4 3 not detected not detected 5.0 95.0 6 not detected notdetected 1.5 98.5 yes 6 48.4 not detected 45.0 6.7 5 no 0 47.8 notdetected 44.7 7.5 1 not detected not detected 22.1 77.9 3 not detectednot detected 15.2 84.8 6 not detected not detected 9.2 90.8 yes 6 47.8not detected 44.7 7.5 10 no 0 59.5 not detected 38.2 2.3 1 not detectednot detected 33.3 66.7 3 not detected not detected 25.3 74.7 6 notdetected not detected 18.7 81.3 yes 6 49.5 not detected 45.9 4.6 20 no 050.5 not detected 46.1 3.4 1 not detected not detected 44.1 55.9 3 notdetected not detected 35.8 64.2 6 not detected not detected 28.6 71.4yes 6 49.5 not detected 46.2 4.3 30 no 0 49.4 not detected 46.7 3.8 15.7 not detected 46.6 47.7 3 9.6 not detected 41.4 48.9 6 2.0 notdetected 37.8 60.2 yes 6 50.1 not detected 46.1 3.8

[0145] TABLE 29 Decomposition efficiency of daidzin family in roastedsoy flour Substrate Heat Reaction concentration treatment time GlycosideAglycon (%) of enzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein2 no 0 30.2 not detected 47.6 22.3 1 not detected not detected 9.2 90.83 not detected not detected not detected 100.0 6 not detected notdetected not detected 100.0 yes 6 46.6 not detected 47.2 6.2 5 no 0 46.1not detected 46.1 7.7 1 not detected not detected 20.9 79.1 3 notdetected not detected 12.5 87.5 6 not detected not detected 6.8 93.2 yes6 46.1 not detected 46.1 7.7 10 no 0 60.1 not detected 38.5 1.4 1 notdetected not detected 30.5 69.5 3 not detected not detected 21.5 78.5 6not detected not detected 14.9 85.1 yes 6 47.5 not detected 47.7 4.8 20no 0 50.3 not detected 46.1 3.7 1 not detected not detected 40.7 59.3 3not detected not detected 32.6 67.4 6 not detected not detected 25.174.9 yes 6 48.6 not detected 47.2 4.2 30 no 0 47.7 not detected 47.6 4.71 not detected not detected 46.4 53.6 3 not detected not detected 42.657.4 6 not detected not detected 34.3 65.7 yes 6 49.0 not detected 47.43.6

[0146] TABLE 30 Decomposition efficiency of glycitin family in soymilkHeat Substrate treatment Reaction concentration of time GlycosideAglycon (%) enzyme (h) glycitin malonylglycitin acetyl-glycitinglycitein 2 no 0 71.8 28.2 not detected not detected 1 not detected notdetected not detected 100.0 3 not detected not detected not detected100.0 6 not detected not detected not detected 100.0 yes 6 51.8 48.2 notdetected not detected 5 no 0 49.4 50.6 not detected not detected 1 notdetected 24.6 not detected 75.4 3 not detected 7.5 not detected 92.5 6not detected not detected not detected 100.0 yes 6 56.7 43.3 notdetected not detected 10 no 0 52.5 41.1 not detected 6.4 1 not detected26.4 not detected 73.6 3 not detected 10.5 not detected 89.5 6 notdetected 4.2 not detected 95.8 yes 6 56.8 37.0 not detected 6.2 20 no 052.4 41.3 not detected 6.3 1 not detected 27.8 not detected 72.2 3 notdetected 9.4 not detected 90.6 6 not detected 5.9 not detected 94.1 yes6 55.5 38.8 not detected 5.7 30 no 0 50.9 43.0 not detected 6.1 1 notdetected 33.1 not detected 66.9 3 not detected 15.9 not detected 84.1 6not detected 7.7 not detected 92.3 yes 6 55.8 37.5 not detected 6.7

[0147] TABLE 31 Decomposition efficiency of genistin family in soymilkSubstrate Heat Reaction concentration treatment time Glycoside Aglycon(%) of enzyme (h) genistin malonylgenistin acetyl-genistin genistein 2no 0 54.5 35.0 not detected 10.5 1 not detected 15.3 not detected 84.7 3not detected 3.5 not detected 96.5 6 not detected 0.0 not detected 100.0yes 6 38.0 49.5 not detected 12.5 5 no 0 31.5 58.2 not detected 10.3 1not detected 24.9 not detected 75.1 3 not detected 6.9 not detected 93.16 not detected 2.1 not detected 97.9 yes 6 37.9 52.5 not detected 9.7 10no 0 32.9 56.5 0.5 10.1 1 not detected 31.5 not detected 68.5 3 notdetected 11.9 not detected 88.1 6 not detected 4.8 not detected 95.2 yes6 37.6 51.2 0.5 10.6 20 no 0 33.3 56.3 0.6 9.9 1 not detected 37.6 notdetected 62.4 3 not detected 15.7 not detected 84.3 6 not detected 10.7not detected 89.3 yes 6 38.6 51.0 0.6 9.8 30 no 0 33.6 55.9 0.6 9.9 1not detected 42.5 not detected 57.5 3 not detected 25.4 not detected74.6 6 not detected 17.1 not detected 82.9 yes 6 38.6 50.0 0.6 10.9

[0148] TABLE 32 Decomposition efficiency of daidzin family in soymilkSubstrate Heat Reaction concentration treatment time Glycoside Aglycon(%) of enzyme (h) daidzin malonyldaidzin acetyl-daidzin daidzein 2 no 053.5 32.2 not detected 14.4 1 not detected 27.5 not detected 72.5 3 notdetected 9.9 not detected 90.1 6 not detected 2.9 not detected 97.1 yes6 39.8 47.0 not detected 13.1 5 no 0 34.2 53.6 not detected 12.2 1 notdetected 34.2 not detected 65.8 3 not detected 15.9 not detected 84.1 6not detected 6.1 not detected 93.9 yes 6 40.5 47.6 not detected 12.0 10no 0 34.6 53.4 not detected 12.0 1 not detected 37.4 not detected 62.6 3not detected 20.5 not detected 79.5 6 not detected 10.7 not detected89.3 yes 6 39.4 47.8 not detected 12.8 20 no 0 34.1 53.6 not detected12.3 1 not detected 41.2 not detected 58.8 3 not detected 21.5 notdetected 78.5 6 not detected 16.4 not detected 83.6 yes 6 39.8 48.1 notdetected 12.1 30 no 0 34.9 53.5 not detected 11.6 1 not detected 43.8not detected 56.2 3 not detected 29.7 not detected 70.3 6 not detected21.9 not detected 78.1 yes 6 40.0 46.9 not detected 13.1

[0149] TABLE 33 Decomposition efficiency of glycitin family inconcentrated soybean protein Substrate Heat Reaction concentrationtreatment time Glycoside Aglycon (%) of enzyme (h) glycitinmalonylglycitin acetylglycitin glycitein 2 no 0 52.2 0.4 35.8 11.6 1 notdetected 0.4 14.3 85.3 3 not detected 0.2 5.3 94.4 6 not detected notdetected 0.0 100.0 yes 6 53.4 0.4 35.0 11.2 5 no 0 52.9 0.4 35.6 11.2 1not detected 0.5 27.1 72.4 3 not detected 0.4 17.8 81.9 6 not detected0.2 12.3 87.4 yes 6 54.0 0.4 34.4 11.3 10 no 0 52.9 0.3 35.7 11.0 1  1.60.4 31.2 66.8 3 not detected 0.3 26.2 73.5 6 not detected 0.3 23.9 75.8yes 6 53.4 0.3 35.2 11.1 20 no 0 53.1 0.2 35.9 10.8 1 13.2 0.3 35.4 51.23 not detected 0.2 33.6 66.2 6 not detected 0.3 30.5 69.2 yes 6 52.8 0.435.8 11.1 30 no 0 57.5 0.4 33.9 8.2 1  2.0 0.6 35.9 61.5 3 not detected0.5 30.7 68.9 6 not detected 0.3 28.3 71.4 yes 6 58.0 0.4 33.4 8.2

[0150] TABLE 34 Decomposition efficiency of genistin family inconcentrated soybean protein Substrate Heat Reaction concentrationtreatment time Glycoside Aglycon (%) of enzyme (h) genistinmalonylgenistin acetylgenistin genistein 2 no 0 50.5 not detected 42.96.6 1  1.2 not detected 20.0 78.7 3 not detected not detected 8.3 91.7 6not detected not detected 3.3 96.7 yes 6 51.6 not detected 41.3 7.0 5 no0 51.2 not detected 43.1 5.8 1 not detected not detected 36.4 63.6 3 notdetected not detected 26.3 73.7 6 not detected not detected 17.8 82.2yes 6 52.2 not detected 41.7 6.2 10 no 0 50.6 not detected 43.4 5.9 1 1.3 not detected 42.8 55.8 3 not detected not detected 37.3 62.7 6 notdetected not detected 33.2 66.8 yes 6 50.9 not detected 42.8 6.3 20 no 050.4 not detected 43.7 5.9 1  6.0 not detected 47.0 46.9 3 not detectednot detected 44.6 55.4 6 not detected not detected 42.3 57.7 yes 6 50.8not detected 43.2 6.1 30 no 0 54.6 not detected 43.5 1.9 1  2.8 notdetected 65.3 31.9 3 not detected not detected 61.0 39.0 6 not detectednot detected 55.9 44.1 yes 6 55.6 not detected 42.4 2.0

[0151] TABLE 35 Decomposition efficiency of daidzin family inconcentrated soybean protein Substrate Heat Reaction concentrationtreatment time Glycoside Aglycon (%) of enzyme (h) diadzin malonydaidzinacetly-daidzin daidzein 2 no 0 52.8 not detected 44.0 3.2 1 not detectednot detected 17.8 82.2 3 not detected not detected  5.3 94.7 6 notdetected not detected not detected 100.0 yes 6 52.6 not detected 43.24.2 5 no 0 53.0 not detected 44.0 3.1 1 not detected not detected 32.667.4 3 not detected not detected 20.5 79.5 6 not detected not detected12.0 88.0 yes 6 53.5 not detected 43.0 3.5 10 no 0 52.8 not detected44.2 3.0 1  2.2 not detected 39.7 58.1 3 not detected not detected 32.967.1 6 not detected not detected 26.9 73.1 yes 6 53.3 not detected 43.43.3 20 no 0 52.9 not detected 44.1 3.0 1  0.7 not detected 46.4 52.9 3not detected not detected 41.5 58.5 6 not detected not detected 37.962.1 yes 6 52.8 not detected 44.0 3.2 30 no 0 49.2 not detected 49.2 1.71  2.7 not detected 56.6 40.7 3 not detected not detected 49.9 50.1 6not detected not detected 42.8 57.2 yes 6 49.8 not detected 48.4 1.8

[0152] TABLE 36 Decomposition efficiency of glycitin family in defattedsoybean Substrate Heat Reaction concentration treatment time GlycosideAglycon (%) of enzyme (h) glycitin malonylglycitin acetylglycitinglycitein 2 no 0 not detected not detected not detected not detected 1not detected not detected not detected not detected 3 not detected notdetected not detected not detected 6 not detected not detected notdetected not detected yes 6 not detected not detected not detected notdetected 5 no 0 not detected 58.4 not detected 41.6 1 not detected 41.6not detected 58.4 3 not detected 31.8 not detected 68.2 6 not detected19.5 not detected 80.5 yes 6 not detected 58.5 not detected 41.5 10 no 0not detected 53.0 not detected 47.0 1 not detected 54.7 not detected45.3 3 not detected 36.6 not detected 63.4 6 not detected 27.9 notdetected 72.1 yes 6 not detected 57.6 not detected 42.4

[0153] TABLE 37 Decomposition efficiency of genistin family in defattedsoybean Substrate Heat Reaction concentration treatment time GlycosideAglycon (%) of enzyme (h) genisitin malonylgenistin acetylgenistingenistein 2 no 0 37.7 45.4 1.8 15.1 1 not detected 31.4 not detected68.6 3 not detected 17.0 not detected 83.0 6 not detected 11.4 notdetected 88.6 yes 6 38.7 41.8 1.5 18.0 5 no 0 40.0 43.9 1.9 14.2 1 notdetected 38.0 0.8 61.2 3 not detected 29.6 0.5 69.9 6 not detected 19.00.3 80.7 yes 6 41.8 41.1 1.8 15.4 10 no 0 42.4 41.6 1.9 14.1 1 notdetected 41.5 1.3 57.2 3 not detected 35.1 0.9 64.0 6 not detected 29.10.6 70.3 yes 6 44.3 39.4 1.7 14.5

[0154] TABLE 38 Decomposition efficiency of daidzin family in defattedsoybean Substrate Heat Reaction concentration treatment time GlycosideAglycon (%) of enzyme (h) daidzin malonyldaidzin acetyldaidzin daidzein2 no 0 42.3 42.2 not detected 15.5 1 not detected 35.2 not detected 64.83 not detected 21.2 not detected 78.8 6 not detected 16.5 not detected83.5 yes 6 43.7 38.1 not detected 18.2 5 no 0 44.0 41.1 not detected14.9 1 not detected 37.7 not detected 62.3 3 not detected 30.6 notdetected 69.4 6 not detected 21.0 not detected 79.0 yes 6 45.8 38.1 notdetected 16.1 10 no 0 45.6 39.7 not detected 14.7 1 not detected 39.5not detected 60.5 3 not detected 34.2 not detected 65.8 6 not detected29.0 not detected 71.0 yes 6 47.5 37.0 not detected 15.5

[0155] By combining maximum reaction temperature and pH, in all thematerials examined, isolation of each isoflavone glucoside was found tobe 70% or more when the material concentration is 10% or less. Inparticular, when the material concentration ranges from 2% to 5%, it wasrevealed that almost 100% conversion of isoflavone glycosides intoaglycons occurred.

EXAMPLE 7 Influence of Commercially Available Enzyme PreparationsAccelerating the Conversion into Aglycon Isoflavones by Diglycosidase

[0156] Soyaflavone (manufactured by Fuji Seiyu K.K.) was suspended into1.0 M sodium acetate of pH 3.0 and the substrate concentration wasadjusted to 30% (w/v) and pH of the solution to 5.0. The suspension waspre-incubated at 50° C. for 1 hour, whereby the temperature of thesuspension was elevated to 50° C. To the suspension was added eachcommercially available enzyme preparation (Amylase AD “Amano” 1, YL-15,Gluczyme NL4.2, Transglucosidase L “Amano”, all manufactured by AmanoEnzyme Inc.) solely or in combination with diglycosidase (0.3 AU) so asto be 0.1% (w/v). The whole was reacted at 50° C. for 6 hours and thechange of the composition of isoflavone glycosides and aglyconisoflavones was analyzed by HPLC. Isoflavone glycosides and aglyconisoflavones were quantitatively determined. Among them, relative valuesof the aglycon isoflavones were shown in FIG. 1, ideal values of theaglycon isoflavones being 100%.

[0157] The ideal value of each aglycon isoflavone was calculated asfollows based on the content of each isoflavone glycoside and aglyconisoflavone in the case that no enzyme was added.

Ideal value of aglycon isoflavone=AG+G1×M _(AG) /M _(G1) +G2×M _(AG) /M_(G2)+ . . .

[0158] AG; amount of aglycon isoflavone, G1; amount of isoflavoneglycoside, M_(AG); molecular weight of aglycon isoflavone, M_(G);molecular weight of isoflavone glycoside

[0159] Each commercially available enzyme preparation it self couldhardly hydrolyze glycosides but the combination with diglycosidaseobviously increased the amount of isolated aglycon isoflavones. Forexample, the combination of diglycosidase with Amylase AD “Amano” 1resulted in 2.1-fold increase of glycitein and 1.3-fold increase ofgenistein as compared with the action of diglycosidase alone. However,about 1.1-fold increase was observed for daidzein. It was revealed thatthe combination of diglycosidase with YL-15 resulted in 1.8-foldincrease of glycitein, 1.2-fold increase of genistein, and 1.1-foldincrease of daidzein, the combination of diglycosidase with GluczymeNL-4.2 resulted in 0.8-fold increase of glycitein, 1.1-fold increase ofgenistein, and 1.0-fold increase of daidzein, and the combination ofdiglycosidase with Transglucosidase L “Amano” resulted in 1.6-foldincrease of glycitein, 1.0-fold increase of genistein, and 1.0-foldincrease of daidzein.

EXAMPLE 8 Examination of Effective Amount of Amylase AD “Amano” 1Accelerating the Conversion into Aglycon Isoflavones by Diglycosidase

[0160] Roasted soy flour (manufactured by Fuji Shokuhin K.K.) wassuspended into 0.1 M sodium acetate adjusted to pH 3.15, the substrateconcentration was adjusted to 30% (w/v), and the pH of the solution to5.0. The suspension was pre-incubated at 50° C. for 1 hour, whereby thetemperature of the suspension was elevated to 50° C. To the suspensionwere added diglycosidase so as to be a final concentration of 0.1% (w/v)(0.3 AU) and Amylase AD “Amano” 1 (manufactured by Amano Enzyme Innc.)so as to be 0.1, 0.05, 0.01, 0.005, and 0.0001% (w/v). The whole wasreacted at 50° C. for 3 hours and the change of isoflavone compositionwas analyzed by HPLC.

[0161] As shown in FIG. 2, it is found that the isolated amount ofaglycon rather increased at a concentration lower than 0.1% (w/v). Aneffect was observed even at a mall amount of 0.0001% (w/v). At 0.005%(w/v), glycitein and genistein increased by a factor of 1.23 and 1.20,respectively. In the case of daidzein, 1.2-fold increase was observed.By the way, 0% means the results in the case of diglycosidase alone.

EXAMPLE 9 Flavor Improvement of Soybean Protein by Diglycosidase or EachCommercially Available Enzyme Preparation

[0162] To 10 g of Fujipro (separated soybean protein, manufactured byFuji Seiyu K.K.) was added 90 mL of water, and the whole was thoroughlymixed to obtain a soybean protein solution. Thereto was addeddiglycosidase or each of commercially available enzyme preparation(Amylase AD “Amano” 1, ADG-S-DS, Lipase A “Amano” 6, Lactase F-DS,Lactase F, Cellulase A “Amano” 3, Hemicellulase “Amano” 90G, Protease B,YL-15, Pectinase PL “Amano”, Transglucosidase L “Amano”, Gluczyme NL4.2,all manufactured by Amano Enzyme Inc.) so that diglycosidase (0.5 AU) oreach enzyme preparation was contained in the soybean protein solution ina concentration of 0.25% (w/v). The treatment was carried out at 50° C.for 5 hours. By the way, the reaction pH was 7.1. For a sensory test, pHwas not adjusted for avoiding the influence of a buffer (the same shallapply to Examples 10 to 12).

[0163] Sensory Test (Flavor Improvement of Separated Soybean ProteinTreated with Diglycosidase or Each Commercially Available EnzymePreparation

[0164] For carrying out a sensory test, each soybean protein treatedwith diglycosidase or each commercially available enzyme preparation wassubjected to centrifugal separation at 1500×g and 4° C. for 20 minutes.The precipitate was removed and pH of the supernatant was adjusted to6.0 with hydrochloric acid. Using a solution obtained by two-folddilution of the solution, the test was carried out. The evaluation wasconducted by five expert panelists, and the deliciousness,bitterness·astringency, and aftertaste were compared with those ofControl untreated with the enzyme (Table 39). TABLE 39 Flavor-improvingeffect of enzyme preparation on separated soybean protein Panelist APanelist B Panelist C bitterness · bitterness · bitterness · Enzyme namedeliciousness astringency aftertaste deliciousness astringencyaftertaste deliciousness astringency aftertaste Untreated (Control) ± ±± ± ± ± ± ± ± Diglycosidase ± −− + ± − + ± −− ± Amylase AD ± −− + ± −− ±± − ± “Amano” 1 ADG-D-DS ± −− + ± − ± ± − ++ Lipase A “Amano” 6 ± ± + ±− + ± ± + Lactase F-DS ± ± ± ± ± − ± ± ± Lactase F “Amano” ± ± ± ± ± ± ±± ± Cellulase A ± −− + ± −− ± ± − ± “Amano” 3 Hemicellulase ± −− + ± −−± ± − ± “Amano” 90G Protease B ± + ± ± ± ± ± ± ± YL-15 ± +++ + ± +++ ± ±+++ ± Pectinase PL ± ± + ± ± ± ± ± ± Trans- glucosidase L ± ± ± ± ± ± ±± ± Gluczyme ± ± ± ± ± ± ± ± ± Panelist D Panelist E bitterness ·bitterness · Enzyme name deliciousness astringency aftertastedeliciousness astringency aftertaste Untreated (Control) ± ± ± ± ± ±Diglycosidase + ± + + − ± Amylase AD “Amano” 1 ± ± ± ± − ± ADG-D-DS +± + ± − ± Lipase A “Amano” 6 ± ± + ± ± ± Lactase F-DS ± ± − ± ± ±Lactase F “Amano” ± ± ± ± ± ± Cellulase A “Amano” 3 + − ± + −− ±Hemicellulase “Amano” 90G ± − ± ± ± + Protease B ± ± ± ± ± ± YL-15 ± +++± ± + ± Pectinase PL ± ± ± ± ± ± Transglucosidase L ± ± ± ± ± ± Gluczyme± ± ± ± ± ±

[0165] As a result, the bitterness-astringency was reduced ordisappeared by diglycosidase, Amylase AD “Amano” 1, ADG-S-DS, Lipase A“Amano” 6, Cellulase A “Amano” 3, or Hemicellulase “Amano” 90G.

[0166] Also, it was revealed that the treatment with diglycosidase,Amylase AD “Amano” 1, ADG-S-DS, Lipase A “Amano” 6, Cellulase A “Amano”3, Hemicellulase “Amano” 90G, YL-15, or Pectinase PL “Amano” waseffective for improving aftertaste. It was revealed that these enzymepreparations improve overall flavor, for example, appearance ofsweetness and reduction of smelling of grass, other than the examinedarticles.

EXAMPLE 10 Flavor Improvement of Soybean Protein by Diglycosidase andEach Commercially Available Enzyme Preparation

[0167] To 10 g of Fujipro (separated soybean protein, manufactured byFuji Seiyu K.K.) was added 90 mL of water, and the whole was thoroughlymixed to obtain a soybean protein solution. Thereto was addeddiglycosidase and each of commercially available enzyme preparation(Amylase AD “Amano” 1, ADG-S-DS, Lipase A “Amano” 6, Lactase F-DS,Lactase F, Cellulase A “Amano” 3, Hemicellulase “Amano” 90G, Protease B,YL-15, Pectinase PL “Amano”, Transglucosidase L “Amano”, Gluczyme NL4.2,all manufactured by Amano Enzyme Inc.) so that diglycosidase wascontained 6.5 AU in the soybean protein solution and each enzymepreparation in a concentration of 0.25% (w/v). The treatment was carriedout at 50° C. for 5 hours to obtain an enzyme-treated soybean protein.By the way, the reaction pH was 7.1.

[0168] Sensory Test (Flavor Improvement of Separated Soybean ProteinTreated with Diglycosidase and Each Commercially Available EnzymePreparation)

[0169] For carrying out a sensory test, each soybean protein treatedwith enzymes was subjected to centrifugal separation at 1500×g and 4° C.for 20 minutes. The precipitate was removed and pH of the supernatantwas adjusted to 6.0 with hydrochloric acid. Using a solution obtained bytwo-fold dilution of the solution, the test was carried out. Theevaluation was conducted by five expert panelists, and thedeliciousness, bitterness·astringency, and aftertaste were compared withthose of Control untreated with the enzyme (Table 40). TABLE 40 Flavorimprovement of separated soybean protein by combination of enzymesPanelist A Panelist B Panelist C bitterness · bitterness · bitterness ·Enzyme name deliciousness astringency aftertaste deliciousnessastringency aftertaste deliciousness astringency aftertaste Untreated(Control) ± ± ± ± ± ± ± ± ± Diglycosidase + Amylase ± −−− + + −−− + ± −−± AD “Amano” 1 Diglycosidase + ADG-S- + −−− ± + − ± ++ −− ++ DSDiglycosidase + Lipase + −−− ± ± − ± + ± + A “Amano”6 Diglycosidase +Lactase ± − ± ± − ± + ± ± F-DS Diglycosidase + Lactase ± ± + + − ± + ± +F “Amano” Diglycosidase + ± −−− ++ + −−− + + − ± Cellulase A “Amano”3Diglycosidase + ± −−− ++ + −− ± + − ± Hemicellulase “Amano”90GDiglycosidase + ± −−− ± + − ± ± −− ± Protease B Diglycosidase + YL-15± + + + + + + ± + Diglycosidase + ± − ++ + − ± + −− ± Pectinase PL“Amano” Diglycosidase + ± − ± ± − ± ± − ± Transglucosidase L “Amano”Diglycosidase + ± ± ± + − ± ± ± ± Gluczyme NL4.2 Panelist D Panelist Ebitterness · bitterness · Enzyme name deliciousness astringencyaftertaste deliciousness astringency aftertaste Untreated (Control) ± ±± ± ± ± Diglycosidase + Amylase AD “Amano”1 ++ ± ± + −− ++Diglycosidase + ADG-S-DS ++ − ++ ± −− + Diglycosidase + Lipase A“Amano”6 ++ ± + ± − + Diglycosidase + Lactase F-DS ± ± ± + − ±Diglycosidase + Lactase F “Amano” + ± + ± −− ++ Diglycosidase +Cellulase A “Amano”3 ++ − ± + −−− + Diglycosidase + Hemicellulase“Amano” 90G ++ − ++ + − ± Diglycosidase + Protease B ++ ± ± ± − +Diglycosidase + YL-15 + ++ + ± ± + Diglycosidase + Pectinase PL “Amano”++ ± + ± − + Diglycosidase + Transglucosidase L “Amano” + ± ± + − ±Diglycosidase + Gluczyme NL4.2 + ± ± ± − ±

[0170] As a result, it was revealed that effects, for example,appearance of sweetness, reduction of bitterness·astringency, orimprovement of aftertaste were observed in all the combinations ofdiglycosidase and each commercially available enzyme preparation(diglycosidase and Amylase AD “Amano” 1, diglycosidase and ADG-S-DS,diglycosidase and Lipase A “Amano” 6, diglycosidase and Lactase F-DS,diglycosidase and Lactase F “Amano”, diglycosidase and Cellulase A“Amano” 3, diglycosidase and Hemicellulase “Amano” 90G, diglycosidaseand Protease B, diglycosidase and YL-15, diglycosidase and Pectinase PL“Amano”, diglycosidase and Transglucosidase L “Amano”, diglycosidase andGluczyme NL4.2) shown in the table. From these facts, it was evidentthat flavor-improving effect was stronger in the combination ofdiglycosidase and each commercially available enzyme preparation than inthe case of diglycosidase alone.

EXAMPLE 11 Flavor Improvement of Soymilk by Diglycosidase or EachCommercially Available Enzyme Preparation

[0171] To 20 mL of an ingredient-unadjusted soymilk (Gitoh ShokuhinK.K.) was added diglycosidase or each of commercially available enzymepreparation (ADG-S-DS, Amylase AD “Amano” 1, Cellulase A “Amano” 3,Hemicellulase “Amano” 90G, all manufactured by Amano Enzyme Inc.) sothat diglycosidase was contained 6.5 AU in the soymilk or each enzymepreparation in a concentration of 0.25% (w/v). The treatment was carriedout at 55° C. for 1.5 hours. Thereafter, the enzymes were inactivated byheat treatment at 70° C. for 1 hour. By the way, the reaction pH was6.6.

[0172] Sensory Test (Flavor Improvement of Soymilk Treated withDiglycosidase or Each Commercially Available Enzyme Preparation)

[0173] The enzyme-treated liquid thus obtained was diluted with water bya factor of 3, and then subjected to a sensory test. The evaluation wasconducted by five expert panelists, and the deliciousness, bitternessastringency, and aftertaste were compared with those of Controluntreated with the enzyme (Table 41). TABLE 41 Flavor-improving effectof enzyme preparation on soymilk Panelist A Panelist B Panelist Cbitter- bitter- bitter- ness* ness* ness* Enzyme sweet- astrin- after-sweet- astrin- after- sweet- astrin- after- name ness gency taste nessgency taste ness gency taste Untreated ± ± ± ± ± ± ± ± ± (Control)Diglycosidase ± − ± ± ± ± + + − + ADG-D-DS + − ± + − + ± − ± AmylaseAD + − − + + − + + − ± “Amano” 1 Cellulase A ± ± ± + − + ± − ± “Amano” 3Hemicellulase ± ± ± + − + ± ± ± “Amano” 90G Panelist D Panelist Ebitter- bitter- ness* ness* Enzyme sweet- astrin- after- sweet- astrin-after- name ness gency taste ness gency taste Untreated ± ± ± ± ± ±(Control) Diglycosidase + ± ± + ± ± ADG-D-DS + ± ± + ± ± Amylase AD ± ±± ± ± ± “Amano” 1 Cellulase A ± ± ± + − + “Amano” 3 Hemicellulase ± ±± + − + “Amano” 90G

[0174] It was revealed that appearance of sweetness, reduction ofbitterness·astringency, and improvement of aftertaste were effected bydiglycosidase or a commercially available enzyme preparation.

EXAMPLE 12 Flavor Improvement of Soymilk by Diglycosidase and EachCommercially Available Enzyme Preparation

[0175] To 20 mL of an ingredient-unadjusted soymilk (Gitoh ShokuhinK.K.) was added diglycosidase and each of commercially available enzymepreparation (ADG-S-DS, Amylase AD “Amano” 1, Cellulase A “Amano” 3, orHemicellulase “Amano” 90G, all manufactured by Amano Enzyme Inc.) sothat diglycosidase was contained 6.5 AU in the soymilk and each enzymepreparation in a concentration of 0.25% (w/v). The treatment was carriedout at 55° C. for 1.5 hours. Thereafter, the enzymes were inactivated byheat treatment at 70° C. for 1 hour. By the way, the reaction pH was6.6.

[0176] Sensory Test (Flavor Improvement of Soymilk Treated withDiglycosidase and Each Commercially Available Enzyme Preparation)

[0177] The enzyme-treated liquid thus obtained was -diluted with waterby a factor of 3, and then subjected to a sensory test. The evaluationwas conducted by five expert panelists, and the deliciousness,bitterness·astringency, and aftertaste were compared with those ofControl untreated with the enzyme (Table 42). TABLE 42 Flavorimprovement of soymilk by combination of enzymes Panelist A Panelist BPanelist C bitter- bitter- bitter- ness* ness* ness* Enzyme sweet-astrin- after- sweet- astrin- after- sweet- astrin- after- name nessgency taste ness gency taste ness gency taste Untreated ± ± ± ± ± ± ± ±± (Control) Diglycosidase + + − − ± + − + + + − − + ADG-S-DSDiglycosidase + + − − − + + − − ± + − − ± Amylase AD “Amano” 1Diglycosidase + ± − ± + − + + + + − − + Cellulase A “Amano” 3Diglycosidase + + − ± + − + + − ± Hemicellulase “Amano” 90G Panelist DPanelist E bitter bitter- ness* ness* Enzyme sweet- astrin- after-sweet- astrin- after- name ness gency taste ness gency taste Untreated ±± ± ± ± ± (Control) Diglycosidase + + − ± + + ± ± ADG-S-DSDiglycosidase + + − + + ± ± Amylase AD “Amano” 1 Diglycosidase + +− + + + − + Cellulase A “Amano” 3 Diglycosidase + + ± ± + + − +Hemicellulase “Amano” 90G

[0178] As a result of the sensory test, it was revealed that effects,for example, appearance of sweetness, reduction ofbitterness·astringency, or improvement of aftertaste were observed inall the combinations of diglycosidase and each commercially availableenzyme preparation (diglycosidase and ADG-S-DS, diglycosidase andAmylase AD “Amano” 1, diglycosidase and Cellulase A “Amano” 3,diglycosidase and Hemicellulase “Amano” 90G) shown in the table.Furthermore, it was revealed that such a treatment with the enzymesimproves overall flavor, for example, reduction of smelling of grass.From these facts, it was evident that flavor-improving effect wasstronger in the combination of diglycosidase and each commerciallyavailable enzyme preparation than in the case of diglycosidase alone ora commercially available enzyme preparation alone.

EXAMPLE 13 Formation of Aglycon Isoflavones from Defatted SoybeanProtein by Diglycosidase at the pH in a Stomach

[0179] Into 20 mM acetate buffer of pH 4.0 was suspended 0.05 g ofdefatted soybean protein (Fuji Seiyu K.K.). The pH of the suspension wasmeasured and the liquid volume was adjusted to 4.5 mL while the pH wasadjusted to 4.0 with 1N hydrochloric acid. An enzyme solution whereinthe diglycosidase activity of crude diglycosidase was adjusted to 1.88AU/mL was added thereto in an amount of 0.5 mL, whereby the final liquidvolume was 5.0 mL (concentration of defatted soybean protein: 1% (w/v)),followed by treatment at 37° C. for 3 hours. After the treatment, 75 μLof methanol and 500 μL of water were added to 25 μL of the reactionmixture. The whole was filtered through a 0.2 μm filter and then thefiltrate was further diluted with water by a factor of 2.5, followed byHPLC analysis.

[0180] As a result of treating defatted soybean protein at pH 4 whichwas the pH range in a stomach during a meal as described above, noisolation of aglycon isoflavones was observed in the treatment at pH 4without adding the enzyme, but isolation of aglycon isoflavones wasobserved in the product treated with diglycosidase. Therefore, it wasproved that diglycosidase could convert isoflavone glycosides intoaglycon isoflavones under the pH condition in a stomach.

EXAMPLE 14 Formation of Aglycon Isoflavones from Roasterd Soy Flour byDiglycosidase at the pH in a Stomach

[0181] Into 100 mL of 50 mM acetate buffer (pH 5) was suspended 2.5 g ofroasted soy flour (manufactured by Kakudai Sangyo).

[0182] To 3 mL of the suspension was added 0.001, 0.002, 0.005, 0.01,0.025, 0.05, 0.075, 0.15, 0.374, 0.75, or 1.5 mg of diglycosidase (290AU/g), followed by incubation under shaking at 37° C. for 30 minutes.After the reaction, 10 mL of methanol was added thereto, and aglyconisoflavones were extracted and analyzed by HPLC.

[0183] As described above, the formation of aglycon isoflavones fromisoflavone glycosides contained in soy flour was investigated after thereaction at pH 5 and 37° C. for 30 minutes on the assumption of theenvironment in a stomach. As a result, as shown in Table 43 and FIG. 3,80% or more of the isoflavone glycosides could be converted into aglyconisoflavones. This result suggests that aglycon isoflavones may be formedfrom isoflavone glycosides in a stomach when diglycosidase is orallyadministered. TABLE 43 Relationship between added amount ofdiglycosidase and formation (%) of aglycon isoflavones aglyconisoflavones diglycosidase (%) 1.500 86.1% 0.750 81.3% 0.374 74.7% 0.15065.4% 0.075 56.3% 0.050 51.1% 0.025 44.3% 0.010 31.0% 0.005 20.3% 0.00212.8% 0.001  9.2% 0  5.0%

EXAMPLE 15 Formation of Aglycon Isoflavones from an IsoflavonePreparation by Diglycosidase at the pH in a Stomach

[0184] Into 100 mL of 50 mM acetate buffer (pH 5) was suspended 150 mgof an isoflavone preparation (manufactured by Nature's Bountry, USA). To3 mL of the suspension was added 0, 0.00045, 0.00113, 0.00225, 0.0045,0.009, 0.0225, 0.045, 0.09, 0.18, 0.45, or 0.9 mg of diglycosidase (290AU/g), followed by incubation under shaking at 37° C. for 30 minutes.After the reaction, 10 mL of methanol was added thereto, and aglyconisoflavones were extracted and analyzed by HPLC.

[0185] As described above, the formation of aglycon isoflavones fromisoflavone glycosides contained in the isoflavone preparation wasinvestigated after the reaction at pH 5 and 37° C. for 30 minutes on theassumption of the environment in a stomach. As a result, as shown inTable 44 and FIG. 4, 80% or more of the isoflavone glycosides could beconverted into aglycon isoflavones. This result suggests that aglyconisoflavones may be formed from isoflavone glycosides in a stomach whendiglycosidase is orally administered. TABLE 44 Relationship betweenadded amount of diglycosidase and formation (%) of aglycon isoflavonesaglycon diglycosidase isoflavones (mg) (%) 0.90000 79.4% 0.45000 78.7%0.18000 75.3% 0.09000 70.7% 0.04500 64.6% 0.02250 59.2% 0.00900 47.5%0.00450 38.9% 0.00225 30.3% 0.00113 25.6% 0.00045 21.5% 0.00000 20.0%

INDUSTRIAL APPLICABILITY

[0186] A physiologically active substance of aglycon type can beefficiently produced, without resort to any acid/alkali treatment orfermentation and substantially without changing the physical propertiesof a material.

[0187] Since diglycosidase has a nature of well acting on 6″-O-acetyland 6″-O-malonylglucosides which are resistant to decomposition byconventional glucosidase, the process can be conducted at one-stepwithout requiring the process of converting decomposition-resistantisoflavone glycosides into isoflavone glycosides decomposable byglucosidase, the process being described in JP-A-10-117792. Moreover,the present process hardly causes change of physical properties of astarting material derived from decomposition of proteins orphospholipids, the decomposition being caused during the process ofhydrolysis with a strong acid. Furthermore, by using diglycosidaseand/or a specific enzyme preparation, the aglycon content in a proteinor protein-containing food can be increased and also the flavor thereofcan be improved.

1. A process for producing an aglycon which comprises forming an aglyconby treating, with diglycosidase, a glycoside containing a compoundselected from the group consisting of phytoestrogens, polyphenols,isoflavones, biochanin A, formononetin, cumestrol, and lignans as theaglycon.
 2. The process for producing an aglycon according to claim 1,wherein the aglycon is an isoflavone.
 3. The process for producing anaglycon according to claim 1 or 2, wherein the glycoside containing anisoflavone as the aglycon is one or more selected from the groupconsisting of daidzin, genistin, or glycitin and acetyl derivatives,succinyl derivatives, or malonyl derivatives thereof.
 4. The process forproducing an aglycon according to any one of claims 1 to 3, wherein thediglycosidase is a glucose-tolerant one.
 5. The process for producing anaglycon according to any one of claims 1 to 4, wherein the diglycosidaseis diglycosidase produced by Penicillium multicolor IAM
 7153. 6. Aprocess for producing a protein having an increased aglycon content or afood containing the protein, which comprises a step of treating aprotein or protein-containing food with diglycosidase.
 7. The processfor producing a protein having an increased aglycon content or a foodcontaining the protein according to claim 6, wherein the protein orprotein-containing food contains a glycoside containing an isoflavone asthe aglycon.
 8. The process for producing a protein having an increasedaglycon content or a food containing the protein according to claim 6 or7, wherein the protein or protein-containing food to be produced is afurther flavor-improved one.
 9. The process for producing a proteinhaving an increased aglycon content or a food containing the proteinaccording to any one of claims 6 to 8, wherein the glycoside containingan isoflavone as the aglycon is one or more selected from the groupconsisting of daidzin, genistin, or glycitin and acetyl derivatives,succinyl derivatives, or malonyl derivatives thereof.
 10. The processfor producing a protein having an increased aglycon content or a foodcontaining the protein according to any one of claims 6 to 9, whichfurther comprises a step of treating with an enzyme preparationcontaining mainly at least one enzyme selected from the group consistingof amylases, proteases, lipases, α-glucosidases, and yeast-dissolvingenzymes.
 11. The process for producing a protein having an increasedaglycon content or a food containing the protein according to any one ofclaims 6 to 10, wherein the improvement of flavor is reduction ofbitterness and/or astringency.
 12. A process for producing aflavor-improved protein or a food containing the protein, whichcomprises a step of treating with an enzyme preparation containingmainly at least one enzyme selected from the group consisting ofamylases, cellulases, pectinases, proteases, lipases, α-glucosidases,α-galactosidases, and yeast-dissolving enzymes.
 13. The process forproducing a flavor-improved protein or a food containing the proteinaccording to claim 12, wherein the protein or protein-containing foodcontains a glycoside containing a flavonoid as the aglycon.
 14. Theprocess for producing a flavor-improved protein or a food containing theprotein according to claim 12 or 13, wherein the protein orprotein-containing food contains a glycoside containing an isoflavone asthe aglycon.
 15. A method of administering diglycosidase orally to forman aglycon from a glycoside in a living body.
 16. The method accordingto claim 15, wherein diglycosidase is orally administered to form anaglycon in a living body from a glycoside containing an isoflavone asthe aglycon.
 17. A method of converting a physiologically activesubstance of glycoside type into a physiologically active substance ofaglycon type, which comprises treating the physiologically activesubstance of glycoside type with diglycosidase.
 18. A process forproducing a composition rich in a phytogenic physiologically activesubstance of aglycon type, which comprises treating a phytogenicmaterial containing a phytogenic physiologically active substance ofglycoside type with diglycosidase.
 19. A method of accelerating abioabsorption of a physiologically active substance, which comprisesadministering diglycosidase orally before, during, or after theingestion of a food containing a physiologically active substance ofglycoside type.
 20. An agent converting a physiologically activesubstance of glycoside type into the physiologically active substance ofaglycon type, which contains at least diglycosidase.